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• W2009068619 abstract "In the yeast Saccharomyces cerevisiae, the transcription of many genes encoding enzymes of phospholipid biosynthesis are repressed in cells grown in the presence of the phospholipid precursors inositol and choline. A genome-wide approach using cDNA microarray technology was used to profile the changes in the expression of all genes in yeast that respond to the exogenous presence of inositol and choline. We report that the global response to inositol is completely distinct from the effect of choline. Whereas the effect of inositol on gene expression was primarily repressing, the effect of choline on gene expression was activating. Moreover, the combination of inositol and choline increased the number of repressed genes compared with inositol alone and enhanced the repression levels of a subset of genes that responded to inositol. In all, 110 genes were repressed in the presence of inositol and choline. Two distinct sets of genes exhibited differential expression in response to inositol or the combination of inositol and choline in wild-type cells. One set of genes contained the UASINO sequence and were bound by Ino2p and Ino4p. Many of these genes were also negatively regulated by OPI1, suggesting a common regulatory mechanism for Ino2p, Ino4p, and Opi1p. Another nonoverlapping set of genes was coregulated by the unfolded protein response pathway, an ER-localized stress response pathway, but was not dependent on OPI1 and did not show further repression when choline was present together with inositol. These results suggest that inositol is the major effector of target gene expression, whereas choline plays a minor role. In the yeast Saccharomyces cerevisiae, the transcription of many genes encoding enzymes of phospholipid biosynthesis are repressed in cells grown in the presence of the phospholipid precursors inositol and choline. A genome-wide approach using cDNA microarray technology was used to profile the changes in the expression of all genes in yeast that respond to the exogenous presence of inositol and choline. We report that the global response to inositol is completely distinct from the effect of choline. Whereas the effect of inositol on gene expression was primarily repressing, the effect of choline on gene expression was activating. Moreover, the combination of inositol and choline increased the number of repressed genes compared with inositol alone and enhanced the repression levels of a subset of genes that responded to inositol. In all, 110 genes were repressed in the presence of inositol and choline. Two distinct sets of genes exhibited differential expression in response to inositol or the combination of inositol and choline in wild-type cells. One set of genes contained the UASINO sequence and were bound by Ino2p and Ino4p. Many of these genes were also negatively regulated by OPI1, suggesting a common regulatory mechanism for Ino2p, Ino4p, and Opi1p. Another nonoverlapping set of genes was coregulated by the unfolded protein response pathway, an ER-localized stress response pathway, but was not dependent on OPI1 and did not show further repression when choline was present together with inositol. These results suggest that inositol is the major effector of target gene expression, whereas choline plays a minor role. Phospholipids are the key structural elements of membrane-bounded organelles and play important roles in signaling and membrane trafficking pathways. Each membrane compartment is composed of a unique set of phospholipids whose biophysical properties contribute to the function of each organelle. Phospholipid metabolism is highly regulated by the cell, ensuring the biogenesis and growth of membranes by coordinating the relative rates of synthesis of individual phospholipids with numerous factors, such as the availability of exogenous supplies of phospholipid precursors, growth stage, and membrane trafficking (1Greenberg M.L. Lopes J.M. Microbiol. Rev. 1996; 60: 1-20Crossref PubMed Google Scholar, 2Carman G.M. Henry S.A. Prog. Lipid Res. 1999; 38: 361-399Crossref PubMed Scopus (264) Google Scholar). In many organisms, the regulation of lipid biosynthetic pathways is achieved through control of transcriptional regulatory networks. For example, lipid homeostasis in animal cells is controlled through the sterol regulatory element binding protein (SREBP) pathway (3Rawson R.B. Nat. Rev. Mol. Cell. Biol. 2003; 4: 631-640Crossref PubMed Scopus (259) Google Scholar). In the budding yeast Saccharomyces cerevisiae, a major control mechanism for the coordinated synthesis of inositoland choline-containing phospholipids is the precise transcriptional control of genes for many of the enzymes required for phospholipid synthesis (2Carman G.M. Henry S.A. Prog. Lipid Res. 1999; 38: 361-399Crossref PubMed Scopus (264) Google Scholar, 4Henry S.A. Patton-Vogt J.L. Prog. Nucleic Acids Res. Mol. Biol. 1998; 61: 133-179Crossref PubMed Google Scholar, 5Carman G.M. Kersting M.C. Biochem. Cell Biol. 2004; 82: 62-70Crossref PubMed Scopus (44) Google Scholar). The synchronized expression of these genes requires the participation of Ino2p and Ino4p, basic helix-loop-helix transcription factors that bind as a heterodimer to a cis-acting element in the promoters of these genes called UASINO, as well as Opi1p, a negative regulator of transcription (6Hoshizaki D.K. Hill J.E. Henry S.A. J. Biol. Chem. 1990; 265: 4736-4745Abstract Full Text PDF PubMed Google Scholar, 7White M.J. Hirsch J.P. Henry S.A. J. Biol. Chem. 1991; 266: 863-872Abstract Full Text PDF PubMed Google Scholar, 8Lopes J.M. Henry S.A. Nucleic Acids Res. 1991; 19: 3987-3994Crossref PubMed Scopus (66) Google Scholar, 9Nikoloff D.M. McGraw P. Henry S.A. Nucleic Acids Res. 1992; 20: 3253Crossref PubMed Scopus (78) Google Scholar, 10Hosaka K. Nikawa J. Kodaki T. Yamashita S. J. Biochem. (Tokyo). 1994; 115: 131-136Crossref PubMed Scopus (21) Google Scholar, 11Nikoloff D.M. Henry S.A. J. Biol. Chem. 1994; 269: 7402-7411Abstract Full Text PDF PubMed Google Scholar, 12Ambroziak J. Henry S.A. J. Biol. Chem. 1994; 269: 15344-15349Abstract Full Text PDF PubMed Google Scholar, 13Bachhawat N. Ouyang Q. Henry S.A. J. Biol. Chem. 1995; 270: 25087-25095Abstract Full Text Full Text PDF PubMed Scopus (106) Google Scholar, 14Schwank S. Ebbert R. Rautenstrauss K. Schweizer E. Schuller H.J. Nucleic Acids Res. 1995; 23: 230-237Crossref PubMed Scopus (109) Google Scholar, 15Wagner C. Dietz M. Wittmann J. Albrecht A. Schuller H.J. Mol. Microbiol. 2001; 41: 155-166Crossref PubMed Scopus (85) Google Scholar). Many of these UASINO-containing genes are maximally derepressed when the phospholipid precursors inositol and choline are absent from the growth medium in logarithmically growing cultures (16Hirsch J.P. Henry S.A. Mol. Cell. Biol. 1986; 6: 3320-3328Crossref PubMed Scopus (181) Google Scholar, 17Bailis A.M. Poole M.A. Carman G.M. Henry S.A. Mol. Cell. Biol. 1987; 7: 167-176Crossref PubMed Scopus (81) Google Scholar, 18Kodaki T. Hosaka K. Nikawa J. Yamashita S. J. Biochem. (Tokyo). 1991; 109: 276-287PubMed Google Scholar, 19Schuller H.J. Hahn A. Troster F. Schutz A. Schweizer E. EMBO J. 1992; 11: 107-114Crossref PubMed Scopus (104) Google Scholar, 20Bailis A.M. Lopes J.M. Kohlwein S.D. Henry S.A. Nucleic Acids Res. 1992; 20: 1411-1418Crossref PubMed Scopus (53) Google Scholar, 21Gaynor P.M. Gill T. Toutenhoofd S. Summers E.F. McGraw P. Homann M.J. Henry S.A. Carman G.M. Biochim. Biophys. Acta. 1991; 1090: 326-332Crossref PubMed Scopus (36) Google Scholar, 22Hasslacher M. Ivessa A.S. Paltauf F. Kohlwein S.D. J. Biol. Chem. 1993; 268: 10946-10952Abstract Full Text PDF PubMed Google Scholar, 23Lai K. McGraw P. J. Biol. Chem. 1994; 269: 2245-2251Abstract Full Text PDF PubMed Google Scholar, 24Shen H. Dowhan W. J. Biol. Chem. 1997; 272: 11215-11220Abstract Full Text Full Text PDF PubMed Scopus (33) Google Scholar, 25Griac P. J. Bacteriol. 1997; 179: 5843-5848Crossref PubMed Google Scholar), suggesting a common regulatory mechanism (4Henry S.A. Patton-Vogt J.L. Prog. Nucleic Acids Res. Mol. Biol. 1998; 61: 133-179Crossref PubMed Google Scholar). A recent collaborative study involving our laboratory has begun to reveal how this regulation is controlled by ongoing lipid metabolism (26Loewen C.J. Gaspar M.L. Jesch S.A. Delon C. Ktistakis N.T. Henry S.A. Levine T.P. Science. 2004; 304: 1644-1647Crossref PubMed Scopus (371) Google Scholar). When inositol is added to the medium of growing cells, a dramatic change to the pattern of phospholipid synthesis is induced. Phosphatidic acid (PA), 1The abbreviations used are: PA, phosphatidic acid; ER, endoplasmic reticulum; UPR, unfolded protein response; YPD, yeast extract/peptone/dextrose; ORF, open reading frame; CDP-DAG, CDP-diacylglycerol; MOPS, 3-(N-morpholino)propane sulfonic acid; PI, phosphatidylinositol; DAG, diacylglycerol; PC, phosphatidylcholine. a precursor for the synthesis of phosphatidylinositol, is rapidly consumed. This drop in PA levels is directly sensed by Opi1p, a component of an endoplasmic reticulum (ER)-localized lipid sensing complex (27Loewen C.J. Roy A. Levine T.P. EMBO J. 2003; 22: 2025-2035Crossref PubMed Scopus (448) Google Scholar), causing it to dissociate from the ER and translocate to the nucleus, where it participates in the repression of target genes. Although Opi1p does not directly interact with the UASINO sequence (28Graves J.A. Henry S.A. Genetics. 2000; 154: 1485-1495PubMed Google Scholar, 29Wagner C. Blank M. Strohmann B. Schuller H.J. Yeast. 1999; 15: 843-854Crossref PubMed Scopus (44) Google Scholar), it is thought to interact with Sin3p (15Wagner C. Dietz M. Wittmann J. Albrecht A. Schuller H.J. Mol. Microbiol. 2001; 41: 155-166Crossref PubMed Scopus (85) Google Scholar), a histone deacetylase that functions as a global transcriptional repressor, and Ino2p, which potentially targets it to the promoters of UASINO-containing genes. Thus, the transcriptional regulation of UASINO-containing genes responds not to inositol directly but instead to a metabolic signal induced when inositol participates in lipid metabolism. Cells grown in the absence of inositol also induce the unfolded protein response (UPR) pathway (30Cox J.S. Chapman R.E. Walter P. Mol. Biol. Cell. 1997; 8: 1805-1814Crossref PubMed Scopus (314) Google Scholar). The UPR pathway is an ER-localized signal transduction pathway that responds to the accumulation of unfolded proteins in the lumen of the ER as well as to secretory stress by up-regulating the expression of target genes (31Cox J.S. Shamu C.E. Walter P. Cell. 1993; 73: 1197-1206Abstract Full Text PDF PubMed Scopus (950) Google Scholar, 32Mori K. Ma W. Gething M.J. Sambrook J. Cell. 1993; 74: 743-756Abstract Full Text PDF PubMed Scopus (655) Google Scholar, 33Mori K. Sant A. Kohno K. Normington K. Gething M.J. Sambrook J.F. EMBO J. 1992; 11: 2583-2593Crossref PubMed Scopus (311) Google Scholar, 34Travers K.J. Patil C.K. Wodicka L. Lockhart D.J. Weissman J.S. Walter P. Cell. 2000; 101: 249-258Abstract Full Text Full Text PDF PubMed Scopus (1599) Google Scholar, 35Chang H.J. Jones E.W. Henry S.A. Genetics. 2002; 162: 29-43Crossref PubMed Google Scholar). Although the mechanism for UPR induction under inositol-limiting conditions is unknown, Cox et al. (30Cox J.S. Chapman R.E. Walter P. Mol. Biol. Cell. 1997; 8: 1805-1814Crossref PubMed Scopus (314) Google Scholar) have suggested that the activation of the UPR might be directly involved in the mechanism by which INO1 transcription is activated. However, recent work from our laboratory has suggested that under certain conditions, UPR activation is not coupled to activation of UASINO-containing genes (35Chang H.J. Jones E.W. Henry S.A. Genetics. 2002; 162: 29-43Crossref PubMed Google Scholar, 36Chang H.J. Jesch S.A. Gaspar M.L. Henry S.A. Genetics. 2004; 168: 1899-1913Crossref PubMed Scopus (57) Google Scholar). Although much study of the regulatory effects of inositol and choline has focused upon the transcription of UASINO-containing genes, their effects on the expression of other genes have been largely unexplored. A recent report (37Santiago T.C. Mamoun C.B. J. Biol. Chem. 2003; 278: 38723-38730Abstract Full Text Full Text PDF PubMed Scopus (70) Google Scholar) examined the combined effects of inositol and choline on genome-wide expression in yeast, but the individual contributions of inositol and choline on gene expression were not explored. Because inositol and choline have distinct effects on different branches of phospholipid biosynthetic pathways (4Henry S.A. Patton-Vogt J.L. Prog. Nucleic Acids Res. Mol. Biol. 1998; 61: 133-179Crossref PubMed Google Scholar) (Fig. 1), an important unanswered question is whether the presence of these phospholipid precursors individually affects the expression of the same set(s) of genes. To identify all of the genes whose expression is regulated by inositol and choline, we carried out a genome-wide study using cDNA microarrays to find genes whose expression is regulated, both independently and jointly, by inositol and choline. We report that inositol and choline affect the expression of different sets of genes. Furthermore, we show that OPI1 primarily regulates genes that were previously shown by Lee et al. (38Lee T.I. Rinaldi N.J. Robert F. Odom D.T. Bar-Joseph Z. Gerber G.K. Hannett N.M. Harbison C.T. Thompson C.M. Simon I. Zeitlinger J. Jennings E.G. Murray H.L. Gordon D.B. Ren B. Wyrick J.J. Tagne J.B. Volkert T.L. Fraenkel E. Gifford D.K. Young R.A. Science. 2002; 298: 799-804Crossref PubMed Scopus (2393) Google Scholar) to contain a bound Ino2p and Ino4p, and these genes are distinct from those genes regulated by the UPR pathway. Our results indicate that inositol, not choline, is the major effector of Ino2p-Ino4p- and UPR-targeted gene expression. Together, these results suggest that distinct pathways contribute to regulate the expression of genes in response to changes in phospholipid metabolism induced by phospholipid precursors. Strains and Media—The wild-type strain BY4742 (MATα, his3Δ1, leu2Δ0, lys2Δ0, ura3Δ0) derived from S288C (39Brachmann C.B. Davies A. Cost G.J. Caputo E. Li J. Hieter P. Boeke J.D. Yeast. 1998; 14: 115-132Crossref PubMed Scopus (2646) Google Scholar) and an isogenic opi1Δ strain (MATα, his3Δ1, leu2Δ0, lys2Δ0, ura3Δ0, opi1::KanMx) in which the OPI1 gene was replaced with KanMx (purchased from Research Genetics) were used for the cDNA microarray experiments. Strains were maintained on yeast extract/peptone/dextrose (YPD) plates (1% yeast extract, 2% bactopeptone, 2% glucose, and 2% agar). All experiments were performed using chemically defined synthetic complete media (16Hirsch J.P. Henry S.A. Mol. Cell. Biol. 1986; 6: 3320-3328Crossref PubMed Scopus (181) Google Scholar, 40Dowd S.R. Bier M.E. Patton-Vogt J.L. J. Biol. Chem. 2001; 276: 3756-3763Abstract Full Text Full Text PDF PubMed Scopus (95) Google Scholar), containing (per liter): 20 g of glucose, 5 g of ammonium sulfate, 1 g of potassium phosphate, 0.5 of g magnesium sulfate, 1 g of sodium chloride, 0.1 g of calcium chloride, 0.5 mg of boric acid, 0.04 mg of cupric sulfate, 0.1 mg of potassium iodide, 0.2 mg of ferric chloride, 0.4 mg of manganese sulfate, 0.2 mg of sodium molybdate, 0.4 mg of zinc sulfate, 2 μg of biotin, 400 μg of calcium pantothenate, 2 μg of folic acid, 400 μg of niacin, 200 μg of p-aminobenzoic acid, 400 μg of pyridoxine hydrochloride, 200 μg of riboflavin, 400 μg of thiamine, 20 mg of adenine sulfate, 20 mg of arginine, 20 mg of histidine, 60 mg of leucine, 230 mg of lysine, 20 mg of methionine, 300 mg of threonine, 20 mg of tryptophan, and 40 mg of uracil. Where indicated, media were supplemented with 75 μmmyo-inositol and/or 1 mm choline. Cell Growth—All cultures were grown in synthetic complete growth medium lacking inositol and choline or containing combinations of inositol and choline as indicated at 30 °C with shaking for at least 12 generations before harvesting at mid-log phase as described below. After overnight growth in indicated media at 30 °C, strains were diluted 1:50 in the indicated media and grown for 5–6 h at 30 °C. 250 ml of media were inoculated at an A600 of 0.01 and allowed to grow at 30 °C until the cultures reached mid-log phase growth (A600 = 0.5). Triplicates were prepared for each growth condition. Cells were harvested by filtration, immediately frozen on a dry ice/ethanol bath, and stored at -80 °C. Filtration was chosen as the method for harvesting cells because the recovery of the INO1 transcript was the most reproducible using this procedure. RNA Isolation and Microarray Analysis—Total RNA was extracted by the high temperature acid phenol method (41Ausubel F.M. Current Protocols in Molecular Biology. J. Wiley, New York2001Google Scholar, 42Kohrer K. Domdey H. Methods Enzymol. 1991; 194: 398-405Crossref PubMed Scopus (507) Google Scholar). For some experiments, total RNA was further purified using the RNeasy RNA purification kit (Qiagen, Inc.), or poly(A) RNA was isolated using oligo dT cellulose as described previously (41Ausubel F.M. Current Protocols in Molecular Biology. J. Wiley, New York2001Google Scholar, 42Kohrer K. Domdey H. Methods Enzymol. 1991; 194: 398-405Crossref PubMed Scopus (507) Google Scholar). mRNA was reverse-transcribed from 20 μg of purified total RNA using 2 μg of oligo dT (18-mer) primers (New England Biolabs) or 2.5 μg of mRNA using 3 μg of random (9-mer) primers (Invitrogen) by incubating with 400 units of SuperScript II (Invitrogen) plus 500 μm concentrations of dATP, dCTP, and dGTP, 200 μm amino-allyl dUTP (Sigma), and 300 μm dTTP in a final volume of 40 μl at 42 °C for 2 h. After cDNA synthesis, the remaining RNA was hydrolyzed by adding 10 μl of both 1 m NaOH and 0.5 m EDTA and incubating at 65 °C for 15 min followed by neutralization with 10 μl of 1 m HCl. The resulting cDNAs were purified using the QIAQuick PCR purification kit (Qiagen, Inc.), substituting kit buffers with KPO4 buffers. Purified cDNAs were labeled with monofunctional reactive Cy3 or Cy5 dye esters (Amersham Biosciences) in the presence of Na2CO3 pH 9.0, for 1 h at room temperature and subsequently quenched with 1 m NH2OH. After an additional QIAQuick purification, 20 pmol of Cy3- and Cy5-labeled cDNA probes were combined and hybridized to Corning CMT Yeast-S288c Gene Arrays (version 1.32) in 5× SSC, 25% formamide, 2.5% SDS, and 100 μg/ml salmon sperm DNA at 42 °C for 14–16 h. After washing, hybridized microarray slides were simultaneously scanned with lasers at 532- and 635-nm bandwidths using a GenePix 4000B array scanner (Axon Instruments, Inc.). Statistical Analysis—Three replicates were analyzed for each experimental condition. Image analysis for each array was processed using the GenePix Pro 4.0 (Axon Instruments, Inc.) software package, which produces (R, G) fluorescence intensity pairs for each gene. After image acquisition, individual data spots on each microarray were visually inspected for size, signal-to-noise ratio, background level, and uniformity. Using these quality control criteria, about 30% of the spots for each triplicate set of experiments were discarded because of poor spot quality, a conventional practice for microarray data analysis (43Wang X. Hessner M.J. Wu Y. Pati N. Ghosh S. Bioinformatics. 2003; 19: 1341-1347Crossref PubMed Scopus (66) Google Scholar). Normalization was conducted as follows: let M = log2(R/G), A = 0.5* log2(R*G). The log ratio M is known to be dependent on overall spot intensity A (44Yang Y.H. Dudoit S. Luu P. Lin D.M. Peng V. Ngai J. Speed T.P. Nucleic Acids Res. 2002; 30: e15Crossref PubMed Scopus (2834) Google Scholar). To remove this systematic variation, intensity-dependent normalization was conducted for each array replicate. The intensity-dependent trend was fitted using the LOWESS fit function (45Dudoit S. Yang Y.H. Callow M.J. Speed T.P. Statistica Sinica. 2002; 12: 111-139Google Scholar) in S-PLUS, after which the log ratio values (M) were normalized by subtracting the trend values. The student t test was performed on normalized M, for each gene for which three high quality replicates were available using the null hypothesis of no change in expression (i.e. normalization to M = 0). A conservative significance level of p value equal to 0.025 corresponding to a false detection rate of q = 0.075 was chosen. Hence the percentage of false positives among the significant tests is controlled to be at most 7.5%, a conservative approach that is expected to reduce false positive identifications to near zero. See Storey (46Storey J.D. J. R. Stat. Soc. Ser. B Stat. Methodol. 2002; 64: 479-498Crossref Scopus (3910) Google Scholar) and Storey and Tibshirani (47Storey J.D. Tibshirani R. Proc. Natl. Acad. Sci. U. S. A. 2003; 100: 9440-9445Crossref PubMed Scopus (7169) Google Scholar) for details on relating adjusted p values to false detection rate and the expected number of false positive tests. The t statistic was computed for each gene, and genes were then ranked according to their p values. Genes were considered to have differential expression in the red channel versus the green channel (or vice versa) if they exhibited a 2-fold change and also had a p value below 0.025. Northern Slot Blot Analysis—Approximately 250 ng of unfractionated mRNA was heated at 65 °C for 15 min in 3 volumes of denaturing buffer (66% formamide, 7% formaldehyde, 26 mm MOPS, 66 mm sodium acetate, and 1.3 mm EDTA), placed on ice after addition of an equal volume of ice-cold 20× SSC, and spotted on BrightStar-Plus (Ambion, Inc.) nylon membranes using a manifold slot blot apparatus as described previously (41Ausubel F.M. Current Protocols in Molecular Biology. J. Wiley, New York2001Google Scholar). Strand-specific 32P-labeled riboprobes were synthesized from plasmids pJH310-INO1 (16Hirsch J.P. Henry S.A. Mol. Cell. Biol. 1986; 6: 3320-3328Crossref PubMed Scopus (181) Google Scholar), pSPACT (36Chang H.J. Jesch S.A. Gaspar M.L. Henry S.A. Genetics. 2004; 168: 1899-1913Crossref PubMed Scopus (57) Google Scholar), pBDG456-Ty1 (kind gift of D. Garfinkel), pBDG458-Ty2 (kind gift of D. Garfinkel), pSJ29-KAR2, pSJ30-PDI1, pSJ31-VTC3 and pSJ32-HO by in vitro transcription according to manufacturer's instructions (Promega). pSJ29-KAR2 was constructed by PCR-amplifying the KAR2 ORF and inserting the 878-bp HindIII-XbaI fragment into pGEM1 (Promega). pSJ30-PDI1 was constructed by PCR-amplifying the PDI1 ORF and inserting the 861-bp HindIII-SalI fragment into pGEM1. pSJ31-VTC3 was constructed by PCR-amplifying the VTC3 ORF and inserting the 703-bp PstI-XbaI fragment into pGEM1. pSJ32-HO was constructed by PCR-amplifying the HO ORF and inserting the 365-bp PstI-BamHI fragment into pGEM1. Membranes were hybridized with either INO1, KAR2, PDI1, VTC3, HO, ACT1, Ty1, or Ty2 probes in formamide hybridization buffer and washed to a final stringency of 0.2× SSC/0.1% SDS at 60 °C as described previously (16Hirsch J.P. Henry S.A. Mol. Cell. Biol. 1986; 6: 3320-3328Crossref PubMed Scopus (181) Google Scholar, 41Ausubel F.M. Current Protocols in Molecular Biology. J. Wiley, New York2001Google Scholar). Quantitation was performed by analysis on a STORM 860 PhosphorImager (Amersham Biosciences) and analyzed with ImageQuant software. The data were normalized by dividing the total counts per minute for the INO1, KAR2, PDI1, VTC3, HO, Ty1, or Ty2 probe by the total counts per minute for the ACT1 probe and expressed as the fraction of the amount of mRNA from cells grown in the absence of inositol and choline. Construction of Gene Disruption Alleles—Complete disruptions of VTC1, VTC3, and VTC4 genes were constructed by PCR-mediated gene replacement as described previously (48Longtine M.S. McKenzie 3rd, A. Demarini D.J. Shah N.G. Wach A. Brachat A. Philippsen P. Pringle J.R. Yeast. 1998; 14: 953-961Crossref PubMed Scopus (4193) Google Scholar) in the wild-type strain BY4742. The plasmid pFA6a-His3MX6 (kind gift of M. Longtine) was used as a template to generate PCR fragments for the gene disruptions. The entire open reading frame of each gene was replaced with the HIS3 marker gene. Histidine prototrophs were screened by colony PCR to verify integration at the correct genetic locus. Assays for Ino-and Opi-Phenotypes—To test for Ino- phenotypes, strains were grown to mid-log phase in synthetic complete media containing 75 μm inositol, washed once with water and 10-fold serial dilutions spotted on plates containing synthetic complete medium lacking inositol. Strains were incubated for 3 days at 18 °C, 24 °C, 30 °C, and 37 °C. To test for Opi- phenotypes (49Greenberg M.L. Reiner B. Henry S.A. Genetics. 1982; 100: 19-33Crossref PubMed Google Scholar, 50Swede M.J. Hudak K.A. Lopes J.M. Henry S.A. Methods Enzymol. 1992; 209: 21-34Crossref PubMed Scopus (15) Google Scholar), strains were grown as described above and spotted on plates containing synthetic complete medium lacking inositol and incubated at 30 °C and 37 °C for 2 days. The plates were then sprayed with a suspension of a diploid tester strain (AID) homozygous for ino1 and ade2 and incubated for an additional 2 days. Global Analysis of Inositol and Choline on Gene Expression in Yeast—Previous studies have shown that the presence of phospholipid precursors in the medium of logarithmically growing cultures of Saccharomyces cerevisiae regulates the transcriptional response of a number of genes. These genes include those encoding the structural enzymes for phospholipid biosynthesis (4Henry S.A. Patton-Vogt J.L. Prog. Nucleic Acids Res. Mol. Biol. 1998; 61: 133-179Crossref PubMed Google Scholar) as well as genes encoding protein folding chaperones induced by the UPR pathway (51Chapman R. Sidrauski C. Walter P. Annu. Rev. Cell Dev. Biol. 1998; 14: 459-485Crossref PubMed Scopus (205) Google Scholar). The goal of the present study was to identify, in a comprehensive manner, the complete set of genes whose expression levels change in response to the presence or absence of the phospholipid precursors inositol and choline using cDNA microarray technology. The cDNA microarray experiments performed in this study are shown in Table I and are summarized below. First, to determine the individual effects of inositol and choline on genome-wide expression, the relative mRNA abundance from cells grown in the presence of either inositol or choline was measured by comparing with transcript levels from cells grown in the absence of both inositol and choline (Table I, experiments 1 and 2). Next, to characterize the combined effects of inositol and choline on genome-wide expression, the relative mRNA abundance from cells grown in the presence of both inositol and choline was compared with transcript levels from cells grown in the absence of inositol with or without choline (Table I, experiments 3 and 4). For every experiment, fluorescently labeled cDNA probes were synthesized from RNA extracted from cells grown to mid-logarithmic phase in chemically defined synthetic media and competitively hybridized to commercial spotted cDNA microarrays containing 6135 unique Saccharomyces cerevisiae open reading frames (ORFs). Each experiment was performed in triplicate. To assess which ORFs showed differential regulation from each experiment, rigorous statistical criteria were used as described under “Experimental Procedures.” In brief, after normalization, the ratio for each spot from the array that showed a p value ≤0.025 over three replicates was considered to be differentially expressed. After statistical analysis, data collected from these experiments were compared and analyzed as described below. The complete data set is available at our laboratory website (www.mbg.cornell.edu/Henry_Lab.cfm).Table IList of experiments performed in this studyNo.Experiment nameExperimental sampleReference sample1Inositol-dependentWild-type I+C-Wild-type I-C-2Choline-dependentWild-type I-C+Wild-type I-C-3Inositol + choline dependent (i)Wild-type I+C+Wild-type I-C-4Inositol + choline dependent (ii)Wild-type I+C+Wild-type I-C+5OPI1 dependent (+ Inositol)opi1Δ I+C-Wild-type I+C-6OPI1 independent (- Inositol)opi1Δ I-C-Wild-type I-C-7opi1Δ controlopi1Δ I+C-opi1Δ I-C- Open table in a new tab In the first experiment (Table I, experiment 1), which measured the independent effects of inositol on genome-wide expression, a total of 32 genes showed a 2-fold or greater change in expression that met our statistical criteria (Fig. 2A). Among these genes, 29 were down-regulated in the presence of inositol compared with cells grown in its absence. These genes are listed in Table II. As expected, the transcript levels of numerous genes previously shown to be affected by inositol levels were down-regulated when inositol was present, including INO1 (16Hirsch J.P. Henry S.A. Mol. Cell. Biol. 1986; 6: 3320-3328Crossref PubMed Scopus (181) Google Scholar), OPI3 (18Kodaki T. Hosaka K. Nikawa J. Yamashita S. J. Biochem. (Tokyo). 1991; 109: 276-287PubMed Google Scholar, 21Gaynor P.M. Gill T. Toutenhoofd S. Summers E.F. McGraw P. Homann M.J. Henry S.A. Carman G.M. Biochim. Biophys. Acta. 1991; 1090: 326-332Crossref PubMed Scopus (36) Google Scholar), ITR1 (23Lai K. McGraw P. J. Biol. Chem. 1994; 269: 2245-2251Abstract Full Text PDF PubMed Google Scholar), and PSD1 (25Griac P. J. Bacteriol. 1997; 179: 5843-5848Crossref PubMed Google Scholar). INO1 showed the greatest difference in expression, consistent with previous reports that this gene is the most highly regulated of the UASINO-containing genes (2Carman G.M. Henry S.A. Prog. Lipid Res. 1999; 38: 361-399Crossref PubMed Scopus (264) Google Scholar). A large number of genes involved in protein folding and secretion, including genes not previously shown to be regulated by inositol, were also down-regulated when inositol was present. We were surprised to find that only three" @default.
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• W2009068619 date "2005-03-01" @default.
• W2009068619 modified "2023-10-16" @default.
• W2009068619 title "Genome-wide Analysis Reveals Inositol, Not Choline, as the Major Effector of Ino2p-Ino4p and Unfolded Protein Response Target Gene Expression in Yeast" @default.
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• W2009068619 doi "https://doi.org/10.1074/jbc.m411770200" @default.
• W2009068619 hasPubMedCentralId "https://www.ncbi.nlm.nih.gov/pmc/articles/1352320" @default.
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