@article {1456, title = {Saccharomyces cerevisiae essential genes with an Opi- phenotype.}, journal = {G3 (Bethesda)}, volume = {4}, year = {2014}, month = {2014 Apr}, pages = {761-7}, abstract = {

The overproduction and secretion of inositol (i.e., Opi(-)) phenotype is associated with defects in regulation of phospholipid biosynthesis in yeast. Here we report a screen of the essential yeast gene set using a conditional-expression library. This screen identified novel functions previously unknown to affect phospholipid synthesis.

}, keywords = {Cluster Analysis, Genes, Essential, Histone Acetyltransferases, Inositol, Phenotype, Phospholipids, Saccharomyces cerevisiae, Saccharomyces cerevisiae Proteins}, issn = {2160-1836}, doi = {10.1534/g3.113.010140}, author = {Salas-Santiago, Bryan and Lopes, John M} } @article {1458, title = {Transcription regulation of a yeast gene from a downstream location.}, journal = {J Mol Biol}, volume = {425}, year = {2013}, month = {2013 Feb 8}, pages = {457-65}, abstract = {

Mechanisms for coregulation of transcription of tandem genes in yeast remain largely speculative. This study focused on inositol-mediated regulation of the tandem gene pair SNA3-INO1. While the pattern of regulation of these two genes was similar, results showed that intermediate levels of inositol repressed INO1 and induced SNA3. Results also showed that inositol-mediated regulation of the SNA3 gene was not a function of its promoter but occurred from factors within the SNA3-INO1 intergenic region. The basic helix-loop-helix proteins, Ino2p and Ino4p, mediated this regulation through the upstream activation sequence (UAS)(INO) (E-box) sequences in the intergenic region. These results provide a model for studying coregulation of yeast tandem genes. This is especially significant given that many tandem gene pairs in yeast are coregulated even though context-specific UAS sequences are known only for one gene in the pair.

}, keywords = {Gene Expression Regulation, Fungal, Inositol, Membrane Proteins, Myo-Inositol-1-Phosphate Synthase, Saccharomyces cerevisiae, Saccharomyces cerevisiae Proteins, Transcription, Genetic}, issn = {1089-8638}, doi = {10.1016/j.jmb.2012.11.018}, author = {Shetty, Ameet and Swaminathan, Aishwarya and Lopes, John M} } @article {32, title = {Transcription regulation of the Saccharomyces cerevisiae PHO5 gene by the Ino2p and Ino4p basic helix-loop-helix proteins.}, journal = {Mol Microbiol}, volume = {83}, year = {2012}, month = {2012 Jan}, pages = {395-407}, abstract = {

The Saccharomyces cerevisiae PHO5 gene product accounts for a majority of the acid phosphatase activity. Its expression is induced by the basic helix-loop-helix (bHLH) protein, Pho4p, in response to phosphate depletion. Pho4p binds predominantly to two UAS elements (UASp1 at -356 and UASp2 at -247) in the PHO5 promoter. Previous studies from our lab have shown cross-regulation of different biological processes by bHLH proteins. This study tested the ability of all yeast bHLH proteins to regulate PHO5 expression and identified inositol-mediated regulation via the Ino2p/Ino4p bHLH proteins. Ino2p/Ino4p are known regulators of phospholipid biosynthetic genes. Genetic epistasis experiments showed that regulation by inositol required a third UAS site (UASp3 at -194). ChIP assays showed that Ino2p:Ino4p bind the PHO5 promoter and that this binding is dependent on Pho4p binding. These results demonstrate that phospholipid biosynthesis is co-ordinated with phosphate utilization via the bHLH proteins.

}, keywords = {Acid Phosphatase, Basic Helix-Loop-Helix Transcription Factors, Chromatin Immunoprecipitation, DNA, Fungal, Enhancer Elements, Genetic, Gene Expression Regulation, Fungal, Inositol, Models, Biological, Phospholipids, Promoter Regions, Genetic, Protein Binding, Saccharomyces cerevisiae, Saccharomyces cerevisiae Proteins, Transcription Factors, Transcription, Genetic}, issn = {1365-2958}, doi = {10.1111/j.1365-2958.2011.07941.x}, author = {He, Ying and Swaminathan, Aishwarya and Lopes, John M} } @article {31, title = {Derepression of INO1 transcription requires cooperation between the Ino2p-Ino4p heterodimer and Cbf1p and recruitment of the ISW2 chromatin-remodeling complex.}, journal = {Eukaryot Cell}, volume = {9}, year = {2010}, month = {2010 Dec}, pages = {1845-55}, abstract = {

The Saccharomyces cerevisiae INO1 gene encodes the structural enzyme inositol-3-phosphate synthase for the synthesis de novo of inositol and inositol-containing phospholipids. The transcription of INO1 is completely derepressed in the absence of inositol and choline (I(-) C(-)). Derepression requires the binding of the Ino2p-Ino4p basic helix-loop-helix (bHLH) heterodimer to the UAS(INO) promoter element. We report here the requirement of a third bHLH protein, centromere-binding factor 1 (Cbf1p), for the complete derepression of INO1 transcription. We found that Cbf1p regulates INO1 transcription by binding to sites distal to the INO1 promoter and encompassing the upstream SNA3 open reading frame (ORF) and promoter. The binding of Cbf1p requires Ino2p-Ino4p binding to the UAS(INO) sites in the INO1 promoter and vice versa, suggesting a cooperative mechanism. Furthermore, Cbf1p binding to the upstream sites was required for the binding of the ISW2 chromatin-remodeling complex to the Ino2p-Ino4p-binding sites on the INO1 promoter. Consistent with this, ISW2 was also required for the complete derepression of INO1 transcription.

}, keywords = {Adenosine Triphosphatases, Basic Helix-Loop-Helix Leucine Zipper Transcription Factors, Basic Helix-Loop-Helix Transcription Factors, Chromatin, Chromatin Assembly and Disassembly, Dimerization, Gene Expression Regulation, Fungal, Myo-Inositol-1-Phosphate Synthase, Promoter Regions, Genetic, Protein Binding, Saccharomyces cerevisiae, Saccharomyces cerevisiae Proteins, Transcription Factors, Transcription, Genetic}, issn = {1535-9786}, doi = {10.1128/EC.00144-10}, author = {Shetty, Ameet and Lopes, John M} } @article {33, title = {Multiple bHLH proteins regulate CIT2 expression in Saccharomyces cerevisiae.}, journal = {Yeast}, volume = {27}, year = {2010}, month = {2010 Jun}, pages = {345-59}, abstract = {

The basic helix-loop-helix (bHLH) proteins comprise a eukaryotic transcription factor family involved in multiple biological processes. They have the ability to form multiple dimer combinations and most of them also bind a 6 bp site (E-box) with limited specificity. These properties make them ideal for combinatorial regulation of gene expression. The Saccharomyces cerevisiae CIT2 gene, which encodes citrate synthase, was previously known to be induced by the bHLH proteins Rtg1p and Rtg3p in response to mitochondrial damage. Rtg1p-Rtg3p dimers bind two R-boxes (modified E-boxes) in the CIT2 promoter. The current study tested the ability of all nine S. cerevisiae bHLH proteins to regulate the CIT2 gene. The results showed that expression of CIT2-lacZ reporter was induced in a rho(0) strain by the presence of inositol via the Ino2p and Ino4p bHLH proteins, which are known regulators of phospholipid synthesis. Promoter mutations revealed that inositol induction required a distal E-box in the CIT2 promoter. Interestingly, deleting the INO2, INO4 genes or the cognate E-box revealed phosphate induction of CIT2 expression. This layer of expression required the two R-boxes and the Pho4p bHLH protein, which is known to be required for phosphate-specific regulation. Lastly, the data show that the Hms1p and Sgc1p bHLH proteins also play important roles in repression of CIT2-lacZ expression. Collectively, these results support the model that yeast bHLH proteins coordinate different biological pathways.

}, keywords = {Basic Helix-Loop-Helix Leucine Zipper Transcription Factors, Basic Helix-Loop-Helix Transcription Factors, Citrate (si)-Synthase, Gene Expression Regulation, Enzymologic, Gene Expression Regulation, Fungal, Inositol, Regulatory Sequences, Nucleic Acid, Saccharomyces cerevisiae, Saccharomyces cerevisiae Proteins, Species Specificity}, issn = {1097-0061}, doi = {10.1002/yea.1757}, author = {Chen, Linan and Lopes, John M} } @article {34, title = {Transcription regulation of the Saccharomyces cerevisiae PIS1 gene by inositol and the pleiotropic regulator, Ume6p.}, journal = {Mol Microbiol}, volume = {70}, year = {2008}, month = {2008 Dec}, pages = {1529-39}, abstract = {In Saccharomyces cerevisiae, transcription of most of the phospholipid biosynthetic genes (e.g. INO1, CHO1, CHO2 and OPI3) is repressed by growth in the presence of inositol and choline and derepressed in their absence. This regulation requires the Ino2p and Ino4p activators and the Opi1p repressor. The PIS1 structural gene is required for the synthesis of the essential lipid phosphatidylinositol. Previous reports show that PIS1 expression is uncoupled from inositol/choline regulation, but is regulated by carbon source, hypoxia and zinc. However, in this study we found that the expression of PIS1 is induced twofold by inositol. This regulation did not require Ino2p and Ino4p, although Ino4p was required for full expression. Ino4p is a basic helix-loop-helix protein that requires a binding partner. Curiously, none of the other basic helix-loop-helix proteins affected PIS1 expression. Inositol induction did require another general regulator of phospholipid biosynthesis, Ume6p. Ume6p was found to be a positive regulator of PIS1 gene expression. Ume6p, and several associated factors, were required for inositol-mediated induction and chromatin immunoprecipitation analysis showed that Ume6p directly regulates PIS1 expression. Thus, we demonstrate novel regulation of the PIS1 gene by Ume6p.}, keywords = {Chromatin Immunoprecipitation, DNA-Binding Proteins, Gene Expression Regulation, Fungal, Histone Deacetylases, Inositol, Repressor Proteins, Saccharomyces cerevisiae, Saccharomyces cerevisiae Proteins, Transcription, Genetic, Transferases (Other Substituted Phosphate Groups)}, issn = {1365-2958}, doi = {10.1111/j.1365-2958.2008.06506.x}, author = {Jani, Niketa M and Lopes, John M} } @article {35, title = {Multiple basic helix-loop-helix proteins regulate expression of the ENO1 gene of Saccharomyces cerevisiae.}, journal = {Eukaryot Cell}, volume = {6}, year = {2007}, month = {2007 May}, pages = {786-96}, abstract = {The basic helix-loop-helix (bHLH) eukaryotic transcription factors have the ability to form multiple dimer combinations. This property, together with limited DNA-binding specificity for the E box (CANNTG), makes them ideally suited for combinatorial control of gene expression. We tested the ability of all nine Saccharomyces cerevisiae bHLH proteins to regulate the enolase-encoding gene ENO1. ENO1 was known to be activated by the bHLH protein Sgc1p. Here we show that expression of an ENO1-lacZ reporter was also regulated by the other eight bHLH proteins, namely, Ino2p, Ino4p, Cbf1p, Rtg1p, Rtg3p, Pho4p, Hms1p, and Ygr290wp. ENO1-lacZ expression was also repressed by growth in inositol-choline-containing medium. Epistatic analysis and chromatin immunoprecipitation experiments showed that regulation by Sgc1p, Ino2p, Ino4p, and Cbf1p and repression by inositol-choline required three distal E boxes, E1, E2, and E3. The pattern of bHLH binding to the three E boxes and experiments with two dominant-negative mutant alleles of INO4 and INO2 support the model that bHLH dimer selection affects ENO1-lacZ expression. These results support the general model that bHLH proteins can coordinate different biological pathways via multiple mechanisms.}, keywords = {Basic Helix-Loop-Helix Transcription Factors, beta-Galactosidase, Chromatin Immunoprecipitation, E-Box Elements, Epistasis, Genetic, Evolution, Molecular, Gene Expression Regulation, Fungal, Genes, Dominant, Models, Biological, Mutation, Phosphopyruvate Hydratase, Promoter Regions, Genetic, Recombinant Fusion Proteins, Saccharomyces cerevisiae, Saccharomyces cerevisiae Proteins}, issn = {1535-9778}, doi = {10.1128/EC.00383-06}, author = {Chen, Meng and Lopes, John M} } @article {37, title = {Genomic analysis of the Opi- phenotype.}, journal = {Genetics}, volume = {173}, year = {2006}, month = {2006 Jun}, pages = {621-34}, abstract = {Most of the phospholipid biosynthetic genes of Saccharomyces cerevisiae are coordinately regulated in response to inositol and choline. Inositol affects the intracellular levels of phosphatidic acid (PA). Opi1p is a repressor of the phospholipid biosynthetic genes and specifically binds PA in the endoplasmic reticulum. In the presence of inositol, PA levels decrease, releasing Opi1p into the nucleus where it represses transcription. The opi1 mutant overproduces and excretes inositol into the growth medium in the absence of inositol and choline (Opi(-) phenotype). To better understand the mechanism of Opi1p repression, the viable yeast deletion set was screened to identify Opi(-) mutants. In total, 89 Opi(-) mutants were identified, of which 7 were previously known to have the Opi(-) phenotype. The Opi(-) mutant collection included genes with roles in phospholipid biosynthesis, transcription, protein processing/synthesis, and protein trafficking. Included in this set were all nonessential components of the NuA4 HAT complex and six proteins in the Rpd3p-Sin3p HDAC complex. It has previously been shown that defects in phosphatidylcholine synthesis (cho2 and opi3) yield the Opi(-) phenotype because of a buildup of PA. However, in this case the Opi(-) phenotype is conditional because PA can be shuttled through a salvage pathway (Kennedy pathway) by adding choline to the growth medium. Seven new mutants present in the Opi(-) collection (fun26, kex1, nup84, tps1, mrpl38, mrpl49, and opi10/yol032w) were also suppressed by choline, suggesting that these affect PC synthesis. Regulation in response to inositol is also coordinated with the unfolded protein response (UPR). Consistent with this, several Opi(-) mutants were found to affect the UPR (yhi9, ede1, and vps74).}, keywords = {Gene Expression Regulation, Fungal, Genes, Fungal, Genes, Reporter, Genome, Fungal, Genomics, Inositol, Lac Operon, Models, Biological, Mutation, Phenotype, Phospholipids, Protein Folding, Repressor Proteins, Saccharomyces cerevisiae, Saccharomyces cerevisiae Proteins}, issn = {0016-6731}, doi = {10.1534/genetics.106.057489}, author = {Hancock, Leandria C and Behta, Ryan P and Lopes, John M} } @article {40, title = {Genomic analysis of PIS1 gene expression.}, journal = {Eukaryot Cell}, volume = {4}, year = {2005}, month = {2005 Mar}, pages = {604-14}, abstract = {The Saccharomyces cerevisiae PIS1 gene is essential and required for the final step in the de novo synthesis of phosphatidylinositol. Transcription of the PIS1 gene is uncoupled from the factors that regulate other yeast phospholipid biosynthetic genes. Most of the phospholipid biosynthetic genes are regulated in response to inositol and choline via a regulatory circuit that includes the Ino2p:Ino4p activator complex and the Opi1p repressor. PIS1 is regulated in response to carbon source and anaerobic growth conditions. Both of these regulatory responses are modest, which is not entirely surprising since PIS1 is essential. However, even modest regulation of PIS1 expression has been shown to affect phosphatidylinositol metabolism and to affect cell cycle progression. This prompted the present study, which employed a genomic screen, database mining, and more traditional promoter analysis to identify genes that affect PIS1 expression. A screen of the viable yeast deletion set identified 120 genes that affect expression of a PIS1-lacZ reporter. The gene set included several peroxisomal genes, silencing genes, and transcription factors. Factors suggested by database mining, such as Pho2 and Yfl044c, were also found to affect PIS1-lacZ expression. A PIS1 promoter deletion study identified an upstream regulatory sequence element that was required for carbon source regulation located downstream of three previously defined upstream activation sequence elements. Collectively, these studies demonstrate how a collection of genomic and traditional strategies can be implemented to identify a set of genes that affect the regulation of an essential gene.}, keywords = {Gene Expression Profiling, Gene Expression Regulation, Fungal, Gene Silencing, Genome, Fungal, Glycerol, Oligonucleotide Array Sequence Analysis, Peroxisomes, Phospholipids, Promoter Regions, Genetic, Recombinant Fusion Proteins, Saccharomyces cerevisiae, Transferases (Other Substituted Phosphate Groups)}, issn = {1535-9778}, doi = {10.1128/EC.4.3.604-614.2005}, author = {Gardocki, Mary E and Bakewell, Margaret and Kamath, Deepa and Robinson, Kelly and Borovicka, Kathy and Lopes, John M} } @article {39, title = {Phosphatidylinositol biosynthesis: biochemistry and regulation.}, journal = {Biochim Biophys Acta}, volume = {1735}, year = {2005}, month = {2005 Jul 15}, pages = {89-100}, abstract = {Phosphatidylinositol (PI) is a ubiquitous membrane lipid in eukaryotes. It is becoming increasingly obvious that PI and its metabolites play a myriad of very diverse roles in eukaryotic cells. The Saccharomyces cerevisiae PIS1 gene is essential and encodes PI synthase, which is required for the synthesis of PI. Recently, PIS1 expression was found to be regulated in response to carbon source and oxygen availability. It is particularly significant that the promoter elements required for these responses are conserved evolutionarily throughout the Saccharomyces genus. In addition, several genome-wide strategies coupled with more traditional screens suggest that several other factors regulate PIS1 expression. The impact of regulating PIS1 expression on PI synthesis will be discussed along with the possible role(s) that this may have on diseases such as cancer.}, keywords = {Eukaryotic Cells, Gene Expression Regulation, Fungal, Mutation, Phenotype, Phosphatidylinositols, Saccharomyces cerevisiae, Saccharomyces cerevisiae Proteins, Transferases (Other Substituted Phosphate Groups)}, issn = {0006-3002}, doi = {10.1016/j.bbalip.2005.05.006}, author = {Gardocki, Mary E and Jani, Niketa and Lopes, John M} } @article {41, title = {Identification of novel dominant INO2c mutants with an Opi- phenotype.}, journal = {Mol Microbiol}, volume = {52}, year = {2004}, month = {2004 Jun}, pages = {1271-80}, abstract = {The INO2 gene of Saccharomyces cerevisiae is required for derepression of the phospholipid biosynthetic genes in response to inositol depletion. Conversely, the OPI1 gene is required for repression in response to inositol supplementation. Results of an in vitro assay have led to a model in which Opi1p interacts with Ino2p. However, there is no in vivo evidence to support this model. Additionally, most of the previously isolated ino2 mutants offer little insight into this model. Here, we report the isolation of a new class of dominant mutations in the INO2 gene, which yield constitutive expression of a target gene (i.e. an Opi(-) mutant phenotype). Two mutations reside in a region of the Ino2p required for interaction with Opi1p in vitro. Three other mutations are at the amino-terminus in a transcriptional activation domain.}, keywords = {Basic Helix-Loop-Helix Transcription Factors, Gene Expression Regulation, Fungal, Inositol, Mutation, Phenotype, Repressor Proteins, Saccharomyces cerevisiae, Saccharomyces cerevisiae Proteins, Transcription Factors}, issn = {0950-382X}, doi = {10.1111/j.1365-2958.2004.04069.x}, author = {Gardenour, Kyle R and Levy, Jamie and Lopes, John M} } @article {42, title = {Expression of the yeast PIS1 gene requires multiple regulatory elements including a Rox1p binding site.}, journal = {J Biol Chem}, volume = {278}, year = {2003}, month = {2003 Oct 3}, pages = {38646-52}, abstract = {The PIS1 gene is required for de novo synthesis of phosphatidylinositol (PI), an essential phospholipid in Saccharomyces cerevisiae. PIS1 gene expression is unusual because it is uncoupled from the other phospholipid biosynthetic genes, which are regulated in response to inositol and choline. Relatively little is known about regulation of transcription of the PIS1 gene. We reported previously that PIS1 transcription is sensitive to carbon source. To further our understanding of the regulation of PIS1 transcription, we carried out a promoter deletion analysis that identified three regions required for PIS1 gene expression (upstream activating sequence (UAS) elements 1-3). Deletion of either UAS1 or UAS2 resulted in an approximately 45\% reduction in expression, whereas removal of UAS3 yielded an 84\% decrease in expression. A comparison of promoters among several Saccharomyces species shows that these sequences are highly conserved. Curiously, the UAS3 element region (-149 to -138) includes a Rox1p binding site. Rox1p is a repressor of hypoxic genes under aerobic growth conditions. Consistent with this, we have found that expression of a PIS1-cat reporter was repressed under aerobic conditions, and this repression was dependent on both Rox1p and its binding site. Furthermore, PI levels were elevated under anaerobic conditions. This is the first evidence that PI levels are affected by regulation of PIS1 transcription.}, keywords = {Anoxia, Base Sequence, Binding Sites, Carbon, Chloramphenicol O-Acetyltransferase, Choline, Chromatography, Thin Layer, Conserved Sequence, DNA, DNA, Complementary, DNA-Binding Proteins, Gene Deletion, Gene Expression Regulation, Fungal, Genes, Reporter, Inositol, Lipid Metabolism, Models, Biological, Models, Genetic, Molecular Sequence Data, Oxygen, Phospholipids, Plasmids, Promoter Regions, Genetic, Protein Binding, Repressor Proteins, RNA, Saccharomyces cerevisiae, Saccharomyces cerevisiae Proteins, Transcription, Genetic, Transferases (Other Substituted Phosphate Groups)}, issn = {0021-9258}, doi = {10.1074/jbc.M305251200}, author = {Gardocki, Mary Elizabeth and Lopes, John M} } @article {43, title = {Opi1p, Ume6p and Sin3p control expression from the promoter of the INO2 regulatory gene via a novel regulatory cascade.}, journal = {Mol Microbiol}, volume = {48}, year = {2003}, month = {2003 May}, pages = {823-32}, abstract = {The INO2 gene of Saccharomyces cerevisiae is required for expression of most of the phospholipid biosynthetic genes. INO2 expression is regulated by a complex cascade that includes autoregulation, Opi1p-mediated repression and Ume6p-mediated activation. To screen for mutants with altered INO2 expression directly, we constructed an INO2-HIS3 reporter that provides a plate assay for INO2 promoter activity. This reporter was used to isolate mutants (dim1) that fail to repress expression of the INO2 gene in an otherwise wild-type strain. The dim1 mutants contain mutations in the OPI1 gene. To define further the mechanism for Ume6p regulation of INO2 expression, we isolated suppressors (rum1, 2, 3) of the ume6Delta mutation that overexpress the INO2-HIS3 gene. Two of the rum mutant groups contain mutations in the OPI1 and SIN3 genes showing that opi1 and sin3 mutations are epistatic to the ume6Delta mutation. These results are surprising given that Ume6p, Sin3p and Rpd3p are known to form a complex that represses the expression of a diverse set of yeast genes. This prompted us to examine the effect of sin3Delta and rpd3Delta mutants on INO2-cat expression. Surprisingly, the sin3Delta allele overexpressed INO2-cat, whereas the rpd3Delta mutant had no effect. We also show that the UME6 gene does not affect the expression of an OPI1-cat reporter. This suggests that Ume6p does not regulate INO2 expression indirectly by regulating OPI1 expression.}, keywords = {Basic Helix-Loop-Helix Transcription Factors, DNA-Binding Proteins, Fungal Proteins, Gene Expression Regulation, Fungal, Genes, Regulator, Genes, Reporter, Genetic Complementation Test, Histone Deacetylases, Methyltransferases, Phenotype, Phospholipids, Promoter Regions, Genetic, Repressor Proteins, Saccharomyces cerevisiae, Saccharomyces cerevisiae Proteins, Transcription Factors}, issn = {0950-382X}, author = {Kaadige, Mohan R and Lopes, John M} }