Regulation of the Yeast INO1 Gene: The Products of the INO2, INO4 and OPI1 Regulatory Genes Are Not Required for Repression in Response to Inositol

Corresponding author: Susan A. Henry, Department of Biological Sciences, Carnegie Mellon University, 4440 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

The ino2, ino4, opi1, and sin3 mutations all affect expression of INO1, a structural gene for inositol-1-phosphate synthase. These same mutations affect other genes of phospholipid biosynthesis that, like INO1, contain the repeated element UAS_INO (consensus 5' CATGTGAAAT 3'). In this study, we evaluated the effects of these four mutations, singly and in all possible combinations, on growth and expression of INO1. All strains carrying an ino2 or ino4 mutation, or both, failed to grow in medium lacking inositol. However, when grown in liquid culture in medium containing limiting amounts of inositol, the opi1 ino4 strain exhibited a level of INO1 expression comparable to, or higher than, the wild-type strain growing under the same conditions. Furthermore, INO1 expression in the opi1 ino4 strain was repressed in cells grown in medium fully supplemented with both inositol and choline. Similar results were obtained using the opi1 ino2 ino4 strain. Regulation of INO1 was also observed in the absence of the SIN3 gene product. Therefore, while Opi1p, Sin3p, and the Ino2p/Ino4p complex all affect the overall level of INO1 expression in an antagonistic manner, they do not appear to be responsible for transmitting the signal that leads to repression of INO1 in response to inositol. Various models for Opi1p function were tested and no evidence for binding of Opi1p to UAS_INO, or to Ino2p or Ino4p, was obtained.

THE INO1 gene of yeast encodes inositol-1-phosphate synthase, the enzyme that catalyzes the rate-limiting step in the synthesis of the eukaryotic phospholipid precursor inositol. INO1 transcription is a sensitive indicator of defects in the cellular transcription apparatus. Mutations in the large subunit of RNA polymerase II (SCAFE et al. 1990 ), the TATA binding protein (ARNDT et al. 1995 ; SHIRRA and ARNDT 1999 ), and in components of the SWI/SNF chromatin remodeling complex (PETERSON and HERSKOWITZ 1992 ; PETERSON et al. 1994 ) all lead to inositol auxotrophy (Ino^- phenotype) due to an inability to activate the INO1 gene. Depletion of the general transcription factor, TFIIA, also impairs INO1 activation (LIU et al. 1999 ). Histone H4 mutations have been characterized that are capable of suppressing the Ino^- phenotype associated with mutations in genes that encode components of the SWI/SNF complex (SANTISTEBAN et al. 1997 ). Additionally, mutations in the SIN3 and UME6 genes lead to high-level INO1 expression and an associated overproduction of inositol (Opi^- phenotype; HUDAK et al. 1994 ; JACKSON and LOPES 1996 ). The SIN3 and UME6 gene products are components of a large complex that contains the RPD3 gene product, a histone deacetylase (KASTEN et al. 1997 ; KADOSH and STRUHL 1998 ; RUNDLETT et al. 1998 ). Compared to wild type, the rpd3 mutant displays an increase of 32-fold in INO1 expression. It also exhibits a 5-fold increase in the acetylation of the lysine 5 residue of histone H4 that is associated with the INO1 promoter (RUNDLETT et al. 1998 ).

In wild-type cells, INO1 is expressed only during the logarithmic phase of growth (JIRANEK et al. 1998 ) and only in the absence of inositol (HIRSCH and HENRY 1986 ). During logarithmic growth, the INO1 gene can be repressed in response to inositol only if synthesis of phosphatidylcholine (PC) is ongoing (GRIAC et al. 1996 ). Excessive turnover of PC via a phospholipase D-mediated route leads to INO1 derepression, even in the presence of inositol and during stationary phase (PATTON-VOGT et al. 1997 ; SREENIVAS et al. 1998 ). These observations have led to the hypothesis that one or more metabolites involved in phospholipid biosynthesis and/or turnover are directly involved in generating the signal(s) for derepression/repression of INO1 (HENRY and PATTON-VOGT 1998 ).

The opi1, ino2, and ino4 mutants were identified on the basis of specific defects in the regulation and/or expression of INO1 and other UAS_INO-containing genes. The ino2 and ino4 mutants were originally isolated as inositol auxotrophs (CULBERTSON and HENRY 1975 ) and were later shown to be pleiotropic, exhibiting deficiencies in PC biosynthesis (LOEWY and HENRY 1984 ), as well as inositol metabolism (CULBERTSON et al. 1976 ). The INO2 and INO4 genes encode basic helix-loop-helix (bHLH) proteins that form a heterodimer and activate transcription by binding to a repeated element (UAS_INO) (consensus: 5' CATGTGAAAT 3') found in the promoter of INO1 and other coregulated genes of phospholipid biosynthesis (AMBROZIAK and HENRY 1994 ; BACHHAWAT et al. 1995 ; SCHWANK et al. 1995 ). The opi1 mutants were originally isolated on the basis of an inositol overproduction (Opi^-) phenotype (GREENBERG et al. 1982 ) and were later shown to express a number of phospholipid biosynthetic enzymes constitutively (KLIG et al. 1985 ). The mechanism of action of the OPI1 gene product, which contains leucine zipper and polyglutamine stretch motifs, is presently unknown (WHITE et al. 1991 ; GRAVES 1996 ; PATTON-VOGT and HENRY 1998 ; WAGNER et al. 1999 ).

A complete understanding of the relative roles of the regulatory genes INO2, INO4, and OPI1 is essential to the elucidation of the overall regulatory network controlling INO1 and other UAS_INO-containing, coregulated genes. In particular, it is essential to gain insight into the mechanism by which the signal for repression in response to inositol is transmitted to the regulatory apparatus of the cell. In this article, we examine the phenotypes of strains carrying the ino4, ino2, opi1, and sin3 mutations relative to growth, INO1 expression, and ability to form DNA protein complexes with the INO1 promoter. We present evidence that the INO1 gene can be repressed in response to inositol in certain genetic backgrounds in which OPI1, INO2, INO4, or SIN3 has been deleted. We conclude that the Ino2p/Ino4p complex, Opi1p, and Sin3p are not required for the regulatory response to inositol. These findings have important implications for our understanding of the mechanism by which repression of UAS_INO-containing genes occurs in response to inositol.

	MATERIALS AND METHODS

TOP ABSTRACT MATERIALS AND METHODS RESULTS DISCUSSION LITERATURE CITED

Yeast strains, media, and growth conditions:
In previous studies, null mutations of the OPI1, INO2, INO4, and SIN3 genes were separately constructed in the W303 MATa or genetic background, producing the strains OP-2, Disr 1D, NUL2, and SH296, respectively (Table 1). These four strains were used in crosses and tetrads were dissected according to the methods of SHERMAN et al. 1978 to generate a set of genetically related strains containing all possible combinations of the four regulatory mutations (Table 1; JAG strains). The genotypes of these strains were verified in part by replica plating on various drop-out media. Regulatory mutations were identified by plate assays for Opi^- and Ino^- phenotypes and confirmed by analysis of PCR reactions of genomic DNA using primers designed to determine whether the individual deletions were present (data not shown).

Unless noted otherwise, strains were grown at 30° in either YEPD (1% yeast extract, 2% bacto-peptone, 2% glucose) or vitamin-defined synthetic complete media, with or without supplements of 10 µM inositol or 75 µM inositol and 1 µM choline, containing 3% glucose, 0.17% yeast nitrogen base salts, 0.5% ammonium sulfate, 0.0002% trace components, and 1% vitamin mix as previously described (GREENBERG et al. 1982 ). Additionally, the following amino acids and supplements were added (milligrams per liter): adenine (20), arginine (20), histidine (20), leucine (60), lysine (230), methionine (20), threonine (300), tryptophan (230), and uracil (20).

Medium supplemented with 75 µM inositol and 1 mM choline is designated I(75)C. The combined presence of inositol and choline at these concentrations has been shown to result in the full repression of transcription of the phospholipid biosynthetic structural genes (HIRSCH and HENRY 1986 ; BAILIS et al. 1987 ; HENRY and PATTON-VOGT 1998 ). Medium supplemented with 10 µM inositol is designated I(10). This concentration of inositol permits an intermediate level of expression of the structural genes, while allowing the inositol auxotrophs to grow (HIRSCH and HENRY 1986 ). Medium completely lacking inositol and choline (designated I^-) was also utilized. This growth condition has been demonstrated to allow complete derepression of the transcription of the phospholipid structural genes, but it does not permit growth of inositol auxotrophs.

To score markers or to select diploids or transformants based on nutritional prototrophies, strains were grown in synthetic medium lacking the appropriate amino acid or other nutrients. Strains were maintained on the appropriate plates containing 2% agar. Potassium acetate plates (0.1% yeast extract, 0.05% glucose, 1% potassium acetate, 2% agar) were used to induce diploids to sporulate.

RNA isolation and analysis:
Total RNA was purified from yeast grown to the middle of the logarithmic growth phase using glass bead disruption and hot phenol extractions (ELION and WARNER 1984 ). RNA was analyzed by the Northern and slot blot assay methods of HIRSCH and HENRY 1986 . The ribosomal gene TCM1 was used as a control for total RNA levels. As previously described (HIRSCH and HENRY 1986 ), TCM1 expression is not influenced by the presence or absence of phospholipid precursors. Quantitation of the blots was performed using the AMBIS (San Diego) imaging system.

To generate the riboprobes used in this study, the appropriate plasmids (Table 2) were linearized (INO1, pJH310 HindIII; TCM1, pAB309 EcoRI; Table 2) and purified by phenol extraction and ethanol precipitation. The DNA was resuspended in 5 µl H₂O for use in the Gemini in vitro transcription system (Promega Corp., Madison, WI). The probes were transcribed with the appropriate polymerase (INO1 T7, TCM1 SP6) in the presence of [-³²P]CTP, which was substituted for unlabeled CTP. The probes were run over a NucTrap column (Stratagene, La Jolla, CA) to remove the unincorporated nucleotides.

Electrophoretic mobility shift assays:
Yeast strains were grown to midlogarithmic phase, and whole cell extracts were prepared as described by LOPES and HENRY 1991 . The protein concentration of extracts was determined using the microassay of the Bio-Rad (Richmond, CA) protein assay kit. Electrophoretic mobility shift assays (EMSAs) were performed according to the methods of LOPES and HENRY 1991 . A fragment of the INO1 promoter (-259 to -154), termed template B, containing two copies of UAS_INO, was prepared as described by LOPES and HENRY 1991 .

A cocktail was made for the entire series of reactions [per reaction: 4 mM Tris/HCl pH 8.0, 4 mM MgCl₂, 4% glycerol, 1 mM dithiothreitol (DTT), 20,000 cpm DNA probe, and 1 µg double-stranded poly(dI-dC) nonspecific competitor]. The experiments employed a final concentration range of 25 mM to 250 mM KCl, with most assays being performed at 50 mM. Once the cocktail had been aliquotted, an aliquot of whole cell extract, containing 50–100 µg of protein, was added. The reactions were incubated at 30° for 15 min and stopped by the addition of 2 µl of 10x dye (0.4% bromophenol blue, 0.4% xylene cyanol, 50% glycerol). Reactions were fractionated on a 4% nondenaturing TBE-polyacrylamide gel, with 1x TBE used as a running buffer.

In vitro transcription/translation:
To perform in vitro transcription (BUTLER and CHAMBERLIN 1982 ; NIELSEN and CHAPIRO 1986 ), 4 µg of the respective plasmids carrying the genes of interest were linearized: OPI1 (pJAG15 NotI), INO2 (pMN103 SalI), and INO4 (pJA755 EcoRI) (Table 2). These complete open reading frames were transcribed with the Promega Gemini transcription kit. The transcripts were phenol extracted and resuspended in sterile water containing pyrocarbonic acid diethyl ester (DEPC). Approximately 4 µg of each in vitro-transcribed RNA was used in in vitro translation reactions using rabbit reticulocyte lysates (Promega; PELHAM and JACKSON 1976 ). The reactions were performed in the presence of ³H-leucine.

The in vitro-translated lysate of interest was diluted ~10-fold in immunoprecipitation buffer (150 mM NaCl, 0.1% NP-40, 50 mM Tris-HCl, pH 8.0). Serum containing polyclonal antibodies directed against Ino2p was added, and the tubes were incubated on ice for 1 hr. The Ino2p antibody was prepared as described in NIKOLOFF and HENRY 1994 . Approximately 4 mg of Protein A-Sepharose bead slurry (Pharmacia, Piscataway, NJ) was added, and the reaction mixture was allowed to incubate at 4° on a rocking bed for at least 1 hr. Samples were centrifuged at 12,000 rpm for 1 min to pellet the immunocomplex. Pellets were washed four times with 100 µl immunoprecipitation buffer and analyzed by gel electrophoresis.

	RESULTS

TOP ABSTRACT MATERIALS AND METHODS RESULTS DISCUSSION LITERATURE CITED

Growth of yeast strains carrying the ino2, ino4, opi1, and sin3 mutations, singly and in combination:
Strains carrying all possible combinations of ino2, ino4, opi1, and/or sin3 mutations (Table 1) were grown to the midlogarithmic phase of growth in YEPD medium, washed, and spotted onto inositol-free (I^-) plates. All strains containing an ino2 or an ino4 mutation, including double and triple mutants containing ino2 and/or ino4 in any combination with opi1 and/or sin3, exhibited no growth after 2 days of incubation at 30° on I^- plates (Fig 1).

View larger version (75K): In this window In a new window Download PPT slide   Figure 1. Growth of phospholipid regulatory mutants on inositol-free (I-) plates. Cells were grown in liquid YEPD medium to midlogarithmic phase, harvested by centrifugation, washed, and spotted at equivalent cell densities onto I- plates. (Top) Growth of strains spotted on an I- plate after 2 days of incubation at 30°. (Bottom) Identification of the strains (full genotypes given in Table 3) as follows: 1, opi1; 2, ino2; 4, ino4; 3, sin3. Strains with multiple mutations are identified as follows: 1/4, opi1 ino4; 1/2/3/4, opi1 ino2 sin3 ino4, etc.

The same strains were then grown in liquid culture under two conditions capable of supporting growth of inositol auxotrophs. The first growth condition consisted of supplementation with 10 µM inositol but no choline. This medium, designated I(10), permits wild-type cells to express INO1 and other UAS_INO-containing genes. However, the level of INO1 expression under this growth condition is lower (i.e., partially depressed) compared to the level of expression observed in wild-type cells growing in medium that is completely inositol free (I^-) (Fig 2, A–C; and HIRSCH and HENRY 1986 ). Partially derepressing, I(10) medium also supports growth of inositol auxotrophs, such as ino2 and ino4 (HIRSCH and HENRY 1986 ). However, the ino2 and ino4 strains both grew more slowly and reached a lower final culture density in I(10) medium than the wild-type strains (Table 3). The ino4 mutant exhibited a doubling time of 4.9 hr in I(10) medium, whereas the ino2 strain essentially failed to grow in I(10) medium. The growth pattern of the ino2 ino4 double mutant in I(10) medium more closely resembled that of the ino4 mutant than that of the ino2 mutant (Table 3).

View larger version (56K): In this window In a new window Download PPT slide   Figure 2. INO1 expression in strains carrying mutations in phospholipid biosynthetic regulatory mutants. Total RNA was harvested from the strains grown to the midlogarithmic phase of growth. The full genotypes of the strains are given in Table 1. The mutations are abbreviated as in Fig 1 as follows: WT, wild type (no mutation); 1, opi1; 2, ino2; 4, ino4; and 3, sin3. The media in which the strains were grown are represented by the following bars: hatched, inositol-free (I-, medium); open, 10 µM inositol [I(10) medium]; and solid, 75 µM inositol and 1 mM choline [I(75)C medium]. The specificity of the riboprobes was verified by Northern blot analysis (data not shown). Subsequently, expression was quantified by slot blot analysis and is presented as a ratio of the counts per minute of the INO1 hybridization to the counts per minute of TCM1 as measured by the AMBIS visualization system. All samples are normalized to the INO1/TMC1 ratio calculated for wild-type cells grown in the absence of inositol. Each data point is representative of three to five experimental repeats. Wild-type INO1 expression is repeated in A–C for purposes of comparison. Note that the scale of B differs from A and C in order to represent the two- to fivefold overexpression of INO1 by opi1- and/or sin3-bearing strains in comparison to wild type. (A) Expression of INO1 in strains containing ino2 and/or ino4 compared to wild type. (B) Strains carrying opi1 and/or sin3 mutations. (C) Strains carrying ino2 and/or ino4 mutations in combination with opi1 and/or sin3.

The second growth condition consisted of supplementation of synthetic complete medium with 75 µM inositol and 1 µM choline (I(75)C medium). Wild-type cells grown in I(75)C medium exhibit full repression of INO1 and other UAS_INO-containing genes (HIRSCH and HENRY 1986 ; BAILIS et al. 1987 ; GRIAC et al. 1996 ). Under these conditions, wild-type cells derive essentially all their inositol from exogenous sources. They also synthesize a substantial proportion of PC via the cytidine diphosphate (CDP)-choline pathway, utilizing exogenous choline (HIRSCH and HENRY 1986 ; GRIAC et al. 1996 ). This fully supplemented growth condition resulted in optimal growth of ino2 and ino4 mutants, permitting them to grow with no apparent deficiency compared to wild type (Table 3). In fact, in I(75)C medium, the ino4 culture achieved a higher final optical density than wild type, whereas the ino2 culture reached a final optical density indistinguishable from the wild-type strain. The ino2 and ino4 strains also exhibited doubling times slightly faster than wild type in I(75)C medium (Table 3). Even wild-type strains exhibit growth stimulation in response to supplementation with inositol and choline (GRIAC et al. 1996 ). In our study, we observed that the wild-type strain doubled every 2.8 hr in I(75)C compared to a doubling time of 3.8 hr in I(10) medium. The wild-type strain also reached a higher final culture density in I(75)C than in I(10) medium (Table 3).

In contrast to the ino2, the ino4, or the wild-type strain, growth of the opi1 strain was not stimulated in I(75)C medium as compared to I(10) medium. In both media, the opi1 strain grew somewhat faster than the wild-type strain did under the most favorable growth condition [i.e., in I(75)C medium]. The opi1 strain also achieved a higher final optical density than wild type in both I(10) and I(75)C medium (Table 3). The fact that the opi1 defect renders the yeast cell constitutive for a high level of expression of enzymes of phospholipid biosynthesis (KLIG et al. 1985 ; WHITE et al. 1991 ; BACHHAWAT et al. 1995 ) may account for the relatively rapid growth of the opi1 strain under inositol-limiting conditions [i.e., I(10) medium, Table 3].

The sin3 strain, like the opi1 strain, exhibited no growth stimulation in I(75)C medium compared to I(10) medium. However, in contrast to the opi1 mutant, the sin3 strain grew significantly slower and achieved a lower optical density than either wild type or opi1 under both growth conditions (Table 3). The fact that the growth of the sin3 strain was not stimulated in I(75)C medium suggests that its growth deficiency is not primarily due to defects in phospholipid metabolism. However, the opi1 sin3 double mutant exhibited a growth rate in both media resembling opi1, suggesting that the elimination of the OPI1 gene product suppresses the relative sin3 growth deficiency. This is somewhat surprising since Sin3p participates in a large protein complex involved in histone deacetylation (KASTEN et al. 1997 ; RUNDLETT et al. 1998 ). This complex has global effects on cellular metabolism and regulation (KADOSH and STRUHL 1998 ; SUN and HAMPSEY 1999 ). In contrast, to date, Opi1p has only been demonstrated to affect lipid metabolism (HENRY and PATTON-VOGT 1998 ).

The opi1 mutation, however, did not restore inositol prototrophy (Fig 1) or alleviate the slow growth phenotype of ino2 and ino4 mutants in I(10) medium. The opi1 ino2, the opi1 ino4, and the opi1 ino2 ino4 strains all failed to grow on I^- medium (Fig 1) and grew more slowly and reached lower optical densities in I(10) medium than the opi1 strain (Table 3). Under fully supplemented conditions [I(75)C medium], the opi1 ino4 double-mutant strain grew somewhat more slowly than either of the single mutants (i.e., opi1 or ino4) but ultimately reached an optical density as high as the opi1 single mutant. The opi1 ino2 strain exhibited a doubling time faster than opi1 ino4 in I(75)C medium and reached a culture density similar to opi1 (Table 3).

The sin3 ino2 double-mutant strain grew faster than the sin3 strain under fully supplemented conditions [I(75)C medium], and it grew as well as the sin3 strain in inositol-limited medium [I(10)]. Thus, the presence of the sin3 mutation, unlike the opi1 mutation, appeared to alleviate the severe ino2 growth deficiency under inositol-limiting conditions [I(10) medium, Table 3]. The sin3 ino4 strain, in contrast, exhibited a growth rate in I(10) medium that was slower than either the ino4 or the sin3 single-mutant strain. The strains containing the triple and quadruple combinations of the four mutations, for the most part, grew more poorly, especially in I(10) medium, than any of the single or double mutant combinations (Table 3).

Expression of the INO1 transcript in strains carrying ino2, ino4, opi1, and sin3 mutations:
Expression of the INO1 transcript was assayed in each strain in the media used for the growth studies described above. The wild-type strain exhibited the expected pattern (HIRSCH and HENRY 1986 ) of INO1 expression and regulation: full derepression in I^- medium and a somewhat lower level of expression (~50% of the level observed in I^- medium) in inositol-limiting medium [I(10)]. Under fully supplemented conditions [I(75)C medium], the level of INO1 transcript in the wild-type strain was ~10% of the level observed in I^- medium (Fig 2, A–C). The expression of INO1 transcript was also examined in the opi1 mutant strain under all three growth conditions [i.e., I^-, I(10), and I(75)C media; Fig 2B]. As previously reported (HIRSCH and HENRY 1986 ; WHITE et al. 1991 ), the INO1 transcript was overexpressed about twofold in the opi1 strain compared to wild type under fully derepressing conditions (I^- medium) and showed essentially no repression in response to the presence of inositol and choline in the growth medium.

The sin3 mutant also overexpressed INO1 transcript severalfold compared to the wild-type strain in I(10) medium. In contrast to the opi1 mutant, however, INO1 expression was repressed in the sin3 strain in response to high levels of inositol and choline [I(75)C medium; Fig 2B], as previously reported (HUDAK et al. 1994 ). The level of INO1 transcript in the opi1 sin3 double mutant strain in I^- medium (Fig 2B) exceeded the levels observed in all other strains studied here, reaching a level fourfold higher than wild type and about twofold higher than in the opi1 strain growing in I^- medium. However, in I(10) and I(75)C medium, INO1 expression in the opi1 sin3 mutant was reduced to a level approximating that observed in the opi1 mutant (i.e., about twofold higher than wild-type derepressed level in I^- medium).

The ino2 and ino4 single-mutant strains both expressed low levels of INO1 transcript in both I(10) and I(75)C medium (Fig 2A), consistent with previous reports (HIRSCH and HENRY 1986 ). In I(75)C medium, INO1 transcript was detected in the ino2ino4 strains at a level slightly higher than that seen in the ino4 strain. However, INO1 transcript was not detected above background when the ino2ino4 strain was grown on I(10) medium (Fig 2A). The opi1ino2 doublemutant strain expressed INO1 transcript at a level only slightly higher than the ino2 single mutant in both I(10) and I(75)C medium, as did the sin3 ino2 strain. The sin3 ino4 strain also expressed very low levels of INO1 transcript under both growth conditions (Fig 2C).

Surprisingly, given its lack of growth on I^- plates (Fig 1), the opi1 ino4 strain, grown under partially derepressing conditions [i.e., I(10) medium], expressed a level of INO1 transcript slightly higher than that seen in the wild-type strain under the same growth conditions. The effects of the opi1 and ino4 mutations on INO1 expression appeared to be additive under partially derepressing conditions [i.e., I(10) medium], since the double mutant exhibited a level of INO1 expression intermediate between the level observed in the two single mutants under these growth conditions (Fig 3, A–C). Moreover, regulation in response to inositol and choline, which is absent in the opi1 mutant, was restored in the opi1 ino4 double mutant, resulting in INO1 repression in I(75)C medium. The opi1 ino2 ino4 triple-mutant strain also expressed a relatively high level of INO1 transcript in I(10) medium, despite its very poor growth in this medium (Table 3), and this expression was repressed in I(75)C medium (Fig 3). The sin3 ino2 ino4 triple mutant also exhibited residual expression in I(10) medium, as did the quadruple mutant opi1 sin3 ino2 ino4, and in both cases INO1 expression was repressed in I(75)C medium. Thus, the elimination of all of these regulatory factors did not completely eliminate INO1 expression or its repression in response to inositol.

View larger version (40K): In this window In a new window Download PPT slide   Figure 3. Electrophoretic mobility shift assays performed with extracts of strains bearing mutations in phospholipid regulatory genes. EMSA reactions were fractionated on 4% nondenaturing gels as described in MATERIALS AND METHODS. Extracts were prepared from cells grown to midlogarithmic phase in YEPD medium. (A) Reactions with aliquots of wild-type extracts containing 50 µg total protein were performed at varying concentrations of KCl: lane 1, 10 mM; lane 2, 25 mM; lane 3, 50 mM; lane 4, 75 mM; lane 5, 100 mM; lane 6, 200 mM. The top arrow indicates the Ino2p/Ino4p complex, and the bottom arrow marks the NBF complex. (B) EMSA reactions were performed in the presence of 50 mM KCl, using cell extracts containing 50 µg of total protein. The extracts correspond to the following lanes: 1, none; 2, wild type; 3, opi1; 4, ino2; 5, ino4; 6, opi1 ino2; 7, opi1 ino4; 8, opi sin3 ino2 ino4; 9, sin3; and 10, 2x wild-type extract (100 µg protein). The middle arrow indicates the Ino2p/Ino4p complex, and the bottom arrow marks the NBF complex. The top arrow indicates a complex that is observed in wild-type (lane 10) and sin3 (not shown) extracts when a higher proportion of protein is used in the reaction, as discussed in RESULTS.

The OPI1 gene product does not appear to bind directly to UAS_INO:
EMSAs were performed using whole-cell extracts of the strains in this study as described in MATERIALS AND METHODS. For these studies, we employed a fragment of the INO1 promoter containing nucleotides -259 to -154 (template B), prepared as described in the MATERIALS AND METHODS. This fragment of DNA contains two copies of UAS_INO (LOPES and HENRY 1991 ) and has been shown to support the formation of a complex with the Ino2p/Ino4p dimer (LOPES and HENRY 1991 ; AMBROZIAK and HENRY 1994 ; NIKOLOFF and HENRY 1994 ), and it is also capable of driving regulated transcription of a heterologous reporter gene (LOPES and HENRY 1991 ).

When wild-type extracts were incubated with template B, two major complexes were detected (Fig 3A), as previously described (LOPES and HENRY 1991 ). Using an oligonucleotide competition assay (data not shown), the complex indicated by the lower arrow (Fig 3A) was identified as the nonamer binding factor (NBF) described by LOPES and HENRY 1991 . The complex migrating directly above the NBF complex (top arrow, Fig 3A; middle arrow, Fig 3B) was absent when ino2 (Fig 3B, lane 4) or ino4 (Fig 3B, lane 5) extracts were used and is therefore identified as the Ino2p/Ino4p complex, as previously described (LOPES and HENRY 1991 ; AMBROZIAK and HENRY 1994 ; NIKOLOFF and HENRY 1994 ).

To determine the optimal binding conditions for the two types of complexes (i.e., Ino2p/Ino4p and NBF), the concentration of potassium chloride in the binding mixture was varied systematically. As the concentration of KCl was increased from 25 mM to 250 mM in binding reactions with wild-type extracts, the intensity of the Ino2p/Ino4p complex was reduced and the intensity of the NBF complex increased (Fig 3A). However, both complexes can be observed at intermediate concentrations such as 50 mM KCl (Fig 3A, lane 3). This intermediate concentration was, therefore, used for subsequent analyses of the mutant strains (Fig 3B). A complex of lower mobility was seen in binding reactions performed with wild-type extracts when a higher concentration of protein was employed (top arrow, Fig 3B, lane 10; compare to lane 2). We believe that this complex is due to separate binding of the Ino2p/Ino4p complex at each of the two UAS_INO sequences in template B, since oligonucleotides containing only one copy of the UAS_INO failed to form the slow migrating complex, no matter how much extract was added (data not shown). Reactions conducted with sin3 extracts contained all of the same bands observed in wild-type reactions (HUDAK et al. 1994 ), as previously reported. As expected, the ino2 (Fig 3B, lane 4) and ino4 (Fig 3B, lane 5) mutants lacked the Ino2p/Ino4p complex and displayed only the NBF complex.

The EMSAs performed with extracts produced from the opi1 strain (Fig 3B, lane 3) produced results similar to those obtained with wild-type extract (Fig 3B, lane 2), except that the levels of both the Ino2/Ino4p complex and NBF appeared to be elevated relative to wild type. However, no complex was observed that was absent in reactions performed with opi1 extracts that was present in reactions carried out with wild-type extract or vice versa. Extracts from all of the double mutants harboring either an ino2 or an ino4 mutation formed the NBF complex, but none of these extracts supported formation of an Ino2p/Ino4p complex (Fig 3B, lanes 6–8, and data not shown). EMSA reactions performed with extracts derived from the opi1 sin3 strain produced results identical to the sin3 mutant extracts (data not shown).

In vitro-cotranslated Ino2p and Ino4p also formed complexes with template B (data not shown), as previously described (AMBROZIAK and HENRY 1994 ). To further assess the possibility that Opi1p might bind UAS_INO directly, in vitro-translated Opi1p was incubated with a fragment of the INO1 promoter (template B) as described in MATERIALS AND METHODS. The in vitro-translated Opi1p failed to form any complex with template B, either alone or in concert with cotranslated Ino2p or Ino4p, and when Opi1p was cotranslated with both Ino2p and Ino4p, only Ino2p/Ino4p complexes were formed (data not shown). Opi1p, Ino2p, and Ino4p cotranslated in vitro were also immunoprecipitated with anti-Ino2p antibody. This analysis reconfirmed the coimmunoprecipitation of Ino4p with Ino2p previously described (AMBROZIAK and HENRY 1994 ). However, Opi1p did not immunoprecipitate with anti-Ino2p when cotranslated with Ino2p, or when Opi1p was cotranslated with Ino2p and Ino4p (data not shown).

	DISCUSSION

TOP ABSTRACT MATERIALS AND METHODS RESULTS DISCUSSION LITERATURE CITED

INO1 is one of the most highly regulated genes in yeast. Not only is its expression repressed some 10- to 30-fold in response to the exogenous phospholipid precursors inositol and choline (HIRSCH and HENRY 1986 ), but it is also regulated in response to growth phase (GRIAC et al. 1996 ; JIRANEK et al. 1998 ). INO1 is also one of a group of apparently unrelated, but highly regulated, genes, the expression of which is commonly found to be affected in mutants having global transcriptional defects (HENRY and PATTON-VOGT 1998 ; SHIRRA and ARNDT 1999 ). Thus, it has become common to check for Ino^- phenotypes in mutants defective in the cellular transcription apparatus (SCAFE et al. 1990 ; SANTISTEBAN et al. 1997 ; LIU et al. 1999 ). The suppression of Ino^- phenotypes has also been used in screens for suppressors of mutants with global transcription defects (SHIRRA and ARNDT 1999 ).

Recently, mutations in several major signal transduction pathways, the unfolded protein response pathway (UPR; COX et al. 1997 ) and the glucose response pathway (OUYANG et al. 1999 ; SHIRRA and ARNDT 1999 ), have also been reported to confer Ino^- or Opi^- phenotypes. The UPR and the glucose response pathways both involve protein phosphorylation and both are required for INO1 expression in a wild-type genetic background. In both cases, the opi1 mutation relieves inositol auxotrophy conferred by mutations (snf1 and ire1) that inactivate protein kinases (Snf1p and Ire1p; COX and WALTER 1996 ; SHIRRA and ARNDT 1999 ). These kinases are required for activation of the glucose response and UPR pathways, respectively.

In contrast to the mutants discussed above, which have defects in the general transcription machinery and/or major cellular signal transduction pathways, the ino2, ino4, and opi1 mutants are believed to be defective primarily in regulation of lipid metabolism (HENRY and PATTON-VOGT 1998 ). However, until this study, no comprehensive assessment of the epistasis of the ino2, ino4, and opi1 mutants had been conducted. An earlier, preliminary study of epistasis involving ino2, ino4, and opi1 point mutants isolated after chemical mutagenesis was limited to a qualitative assessment of Ino^- and Opi^- plate phenotypes (LOEWY et al. 1986 ). The opi1 ino2 and opi1 ino4 strains studied here failed to grow on I^- plates (Fig 1), as previously reported for point mutants (LOEWY et al. 1986 ). This observation is also consistent with the failure of opi1 mutants to be isolated as suppressors of ino4-8 (OUYANG et al. 1999 ) and with the relatively slow growth of the opi1 ino4, opi1 ino2, and opi1 ino2 ino4 strains in inositol-limiting I(10) medium (Table 3).

Whereas Ino2p and Ino4p are known to bind as a heterodimer to UAS_INO (AMBROZIAK and HENRY 1994 ; SCHWANK et al. 1995 ), at present neither the precise role nor the mechanism of Opi1p function is known (GRAVES 1996 ; WAGNER et al. 1999 ). One possible explanation for the constitutive overproduction of inositol (Opi^-) phenotype of the opi1 mutant could be that Opi1p interacts with either one or both of the positive regulators, Ino2p or Ino4p, preventing them from binding to each other and/or to UAS_INO. Alternatively, Opi1p might bind to UAS_INO directly, blocking access to the element by the Ino2p/Ino4p complex. However, the data presented in this report do not support either of these models. We obtained no evidence for direct interaction of Opi1p with Ino2p or Ino4p. Both Ino2p and Ino4p have been shown to be present in a DNA protein complex that binds to UAS_INO-containing DNA fragments (AMBROZIAK and HENRY 1994 ; NIKOLOFF and HENRY 1994 ; BACHHAWAT et al. 1995 ; SCHWANK et al. 1995 ) and this complex is clearly identifiable in the experiments reported here (Fig 3B). However, no Opi1p-dependent DNA-protein complex was observed when extracts of wild-type cells were incubated with an INO1 promoter fragment (Fig 3B). Furthermore, no complex present in the wild-type strain was observed to be absent in the opi1 mutant (Fig 3B). These results are consistent with those of WAGNER et al. 1999 , who detected no evidence for Opi1p binding to DNA or to Ino2p or Ino4p. They did observe that overproduction of Opi1p from a GAL promoter caused yeast strains to become auxotrophic for inositol, consistent with the role of Opi1p as a negative regulator. However, the mechanism of Opi1p function remains elusive.

Consistent with the growth phenotypes observed here (Fig 1, Table 3), there was very little INO1 expression in ino2 and ino4 strains (Fig 2A) in either I(10) or I(75)C medium. Deletion of Opi1p had only a modest effect on residual INO1 expression in the ino2 genetic background (Fig 2C). However, INO1 expression in the opi1 ino4 strain, grown in I(10) medium, was quite high relative to wild type. The expression of INO1 transcript in the opi1 ino4 strain did not seem to be dependent on Ino2p, since INO1 expression was only slightly reduced in the opi1 ino2 ino4 strain compared to the opi1 ino4 strain (Fig 2C), even though this strain grew even more poorly in I(10) medium than did the opi1 ino4 strain (Table 3).

The results reported here reveal subtle differential effects of the ino2 and ino4 mutations on growth and INO1 expression, especially in the opi1 genetic background, suggesting that Ino2p and Ino4p may have some functions distinct from their common role as pairing partners in the Ino2p/Ino4p heterodimer. It has already been demonstrated that these two regulatory genes are differentially regulated. Both the INO2 and OPI1 genes are regulated by the presence of inositol and choline in a manner analogous to INO1 (ASHBURNER and LOPES 1995A , ASHBURNER and LOPES 1995B ; JIRANEK et al. 1998 ), whereas INO4 is constitutively expressed (SCHULLER et al. 1992 ; ASHBURNER and LOPES 1995A ). Moreover, the levels of Ino2p appear to be limiting for the formation of the Ino2p/Ino4p heterodimer (NIKOLOFF and HENRY 1994 ), which binds to UAS_INO, whereas the level of Ino4p does not appear to be limiting for INO1 expression (ASHBURNER and LOPES 1995A , ASHBURNER and LOPES 1995B ). In addition, Ino2p contains an activation domain, while Ino4p does not (SCHWANK et al. 1995 ). A previous study of the effects of ino2 and ino4 point mutants showed greater residual expression of CHO1 (the UAS_INO-containing structural gene for phosphatidylserine synthase) in ino4 mutants than in ino2 mutants (BAILIS et al. 1992 ). Furthermore, ino4 strains can utilize glycerolphosphoryl inositol as a source of inositol, whereas ino2 strains cannot (PATTON-VOGT and HENRY 1998 ). In all of the above-cited instances, ino2 mutants are observed to have more severe defects than ino4 mutants. If Ino2p retained any residual ability to bind to UAS_INO elements in the absence of Ino4p, this could explain the more severe phenotypes of the ino2 mutant. However, no evidence of Ino2p binding in the absence of Ino4p was detected in vitro (AMBROZIAK and HENRY 1994 ; and data not shown). In addition, this hypothesis is not consistent with the observed residual INO1 expression in the opi1 ino2 ino4 strain reported here (Fig 2C). Thus, the residual INO1 expression in the opi1 ino4 genetic background does not seem to be due simply to the presence of the Ino2p activator.

It is possible that transcription factors, other than Ino2p and Ino4p, could be involved in the expression of UAS_INO-containing genes. In a study that may have relevance to this discussion, COK et al. 1998 studied INO2 and INO4 expression in a mutant, nmt1-451D, which has a temperature-sensitive defect in protein myristoylation. The INO2 transcript was found to be elevated, while the INO4 transcript was lower, in nmt1-451D cells in both the presence and absence of inositol. COK et al. 1998 found that expression of the UAS_INO-containing FAS1 gene (fatty acid synthase) is Ino2p dependent in nmt1-451D cells growing at their permissive temperature. However, FAS1 expression is Ino2p independent at the nmt1-451D restrictive temperature where protein myristoylation becomes limiting for growth. COK et al. 1998 suggest that another as-yet-unidentified transcription factor might replace Ino2p function, at least with respect to FAS1 expression, under the circumstances they analyzed.

The residual INO1 transcript expressed in the opi1 ino4 and opi1 ino2 ino4 strains under inositol-limiting conditions was also observed to be regulated in response to inositol and choline (Fig 2C). Thus, at the level of INO1 expression, ino4 is not epistatic to opi1. Rather, the double mutant appears to have a pattern of gene expression closer to wild type than to either single mutant. The opi1 ino2 ino4 strain, and even the opi1 sin3 ino2 ino4 quadruple-mutant strain, exhibited patterns of INO1 expression and regulation similar to the opi1 ino4 strain. Indeed, in all of the strains, other than the opi1 single mutant in which INO1 was expressed at significant levels in I(10) medium, repression was observed in I(75)C medium. The fact that significant regulated INO1 expression was observed in the opi1 ino4, opi1 ino2 ino4, and other ino2 and ino4-bearing strains leads to the question as to why these strains fail to grow on I^- plates and also why they grow quite poorly in I(10) medium. Such slow growth under inositol-limiting conditions is not due simply to failure to express INO1, since the opi1 ino4 strain expressed substantial levels of INO1 transcript (Fig 3C). It also grew more slowly than either single mutant (i.e., opi1 or ino4) under fully supplemented conditions, i.e., in I(75)C medium, suggesting that the opi1 and ino4 mutations may have synergistic negative effects upon processes outside lipid metabolism.

The substantial residual, regulated INO1 expression, which is observed in opi1 ino4 and opi1 ino2 ino4 strains, is surprising and has major implications for any model that purports to explain the regulation of INO1 and other UAS_INO-containing genes in response to inositol and choline. INO1 expression is constitutive when OPI1 is deleted in an otherwise wild-type genetic background (HIRSCH and HENRY 1986 ; WHITE et al. 1991 ). Nevertheless, the results obtained in our study show that Opi1p is not required, at least in an ino4 or an ino2 ino4 genetic background, for the transmission of the signal that leads to repression of INO1 in response to inositol and choline. In addition to observing repression of INO1 in response to inositol and choline in opi1 ino4 and opi1 ino4 ino2 strains, we observed severalfold repression of INO1 expression in the opi1 sin3 and opi1 sin3 ino2 ino4 strains (Fig 2C). Thus, Sin3p is also not required for the transmission of the inositol-responsive signal, but it does affect overall levels of INO1 expression in both OPI1 and opi1 genetic backgrounds. Furthermore, since substantial regulated expression of the INO1 gene is observed in the opi1 ino4 and the opi1 ino2 ino4 strains, we are forced to conclude that the Ino2p/Ino4p complex is not essential for repression of INO1 in response to inositol. The above conclusions are entirely unexpected since the Ino2p/Ino4p heterodimer clearly binds directly to UAS_INO (AMBROZIAK and HENRY 1994 ; SCHWANK et al. 1995 ) and both Ino2p and Ino4p are required for significant expression of INO1 in a wild-type (i.e., OPI1) genetic background (Fig 2) (HIRSCH and HENRY 1986 ). We speculate that Opi1p could be responsible for maintaining a cellular condition conducive to selective binding of Ino2p/Ino4p at UAS_INO, but that neither Opi1p nor the Ino2p/Ino4p complex is responsible for transmitting the signal that leads to repression of UAS_INO-containing genes in response to inositol.

We propose that when Opi1p is eliminated but Ino2p and Ino4p are both present, the Ino2p/Ino4p heterodimer gains enhanced access to UAS_INO elements. Since Ino2p is a highly potent activator (SCHWANK et al. 1995 ), the level of transcription that is sustained under these conditions could "swamp" an underlying regulatory mechanism that is required for repression in response to inositol. Under this scenario, when Ino2p and Ino4p are eliminated along with Opi1p, access to UAS_INO-containing promoters could still be enhanced and would allow access by other less efficient activators. INO1 expression in the strains lacking Opi1p, Ino4p, and/or Ino2p in this case would be due to the effects of transcription factors other than the Ino2p/Ino4p complex having access to the INO1 promoter. The first six nucleotides of the core sequence of UAS_INO (i.e., 5' CATGTG 3') is a fairly generic binding site for proteins of the bHLH family. There are a number of transcription factors in yeast in the bHLH class, in addition to Ino2p/Ino4p, which could potentially recognize such a binding site (BACHHAWAT et al. 1995 ). Alternatively, the as-yet-unidentified NBF (Fig 3) could play a role in basal INO1 expression. NBF is the only complex observed when binding reactions are performed with ino2 or ino4 extracts, whether Opi1p is present or not (Fig 3B).

Whatever activators are responsible for INO1 transcription in the absence of the Ino2p/Ino4p complex in the opi1 genetic background (i.e., in the opi1 ino4 and the opi1 ino2 ino4 strains), it seems likely that the INO1 promoter becomes much more accessible to the basal transcription machinery in the absence of Opi1p. This hypothesis is consistent with the results of SHIRRA and ARNDT 1999 , who demonstrated that deletion of OPI1 results in substantially enhanced INO1 transcription in the spt15-328 mutant, which has a partially defective TATA binding protein. SHIRRA and ARNDT 1999 also observed repression of INO1 in response to inositol in opi1 spt15-328 strains. This result is consistent with our conclusion that Opi1p is not required for the regulatory response to inositol. Whatever the explanation for the inositol/choline-regulated INO1 expression in opi1 spt15-328, opi1 ino2 ino4, and opi1 ino4 strains, we conclude that neither Opi1p nor the Ino2p/Ino4p complex is primarily responsible for transmitting the signal from lipid biosynthesis to the transcription machinery controlling UAS_INO-containing genes. Rather, it appears that Opi1p and the Ino2p/Ino4p complex work antagonistically to attenuate the overall level of expression of UAS_INO-containing genes. Thus, some other as-yet-unidentified factor or condition associated with the transcription of UAS_INO-containing promoters must be responsible for regulating the relative level of transcription in response to the signal produced by the presence of inositol and choline.

	ACKNOWLEDGMENTS

We gratefully acknowledge technical assistance provided by Vincent Bruno. We are especially grateful to Dr. Chi Van Dang of Johns Hopkins University, who provided the opportunity for J.A.G. to complete this manuscript. We are also indebted to our colleagues, Susan R. Dowd, Jana L. Patton-Vogt, Margaret K. Shirra, and Karen M. Arndt for their valuable discussions and criticisms of this manuscript. This work was supported by a National Institutes of Health (NIH) National Research Service Award to J.A.G. (GM-15972) and NIH grant GM-19629 to S.A.H.

Manuscript received August 5, 1999; Accepted for publication December 20, 1999.

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				A. Sreenivas and G. M. Carman Phosphorylation of the Yeast Phospholipid Synthesis Regulatory Protein Opi1p by Protein Kinase A J. Biol. Chem., May 30, 2003; 278(23): 20673 - 20680. [Abstract] [Full Text] [PDF]

				S. Kagiwada and R. Zen Role of the Yeast VAP Homolog, Scs2p, in INO1 Expression and Phospholipid Metabolism J. Biochem., April 1, 2003; 133(4): 515 - 522. [Abstract] [Full Text] [PDF]

				J. Oshiro, S. Rangaswamy, X. Chen, G.-S. Han, J. E. Quinn, and G. M. Carman Regulation of the DPP1-encoded Diacylglycerol Pyrophosphate (DGPP) Phosphatase by Inositol and Growth Phase. INHIBITION OF DGPP PHOSPHATASE ACTIVITY BY CDP-DIACYLGLYCEROL AND ACTIVATION OF PHOSPHATIDYLSERINE SYNTHASE ACTIVITY BY DGPP J. Biol. Chem., December 22, 2000; 275(52): 40887 - 40896. [Abstract] [Full Text] [PDF]

				A. Sreenivas, M. J. Villa-Garcia, S. A. Henry, and G. M. Carman Phosphorylation of the Yeast Phospholipid Synthesis Regulatory Protein Opi1p by Protein Kinase C J. Biol. Chem., August 3, 2001; 276(32): 29915 - 29923. [Abstract] [Full Text] [PDF]

				L. Block-Alper, P. Webster, X. Zhou, L. Supekova, W. H. Wong, P. G. Schultz, and D. I. Meyer IN02, A Positive Regulator of Lipid Biosynthesis, Is Essential for the Formation of Inducible Membranes in Yeast Mol. Biol. Cell, January 1, 2002; 13(1): 40 - 51. [Abstract] [Full Text] [PDF]