Multiple Positive and Negative Elements Involved in the Regulation of Expression of GSY1 in Saccharomyces cerevisiae

GSY1 is one of the two genes encoding glycogen synthase in Saccharomyces cerevisiae. Both the GSY1 message and the protein levels increased as cells approached stationary phase. A combination of deletion analysis and site-directed mutagenesis revealed a complex promoter containing multiple positive and negative regulatory elements. Expression of GSY1 was dependent upon the presence of a TATA box and two stress response elements (STREs). Expression was repressed by Mig1, which mediates responses to glucose, and Rox1, which mediates responses to oxygen. Characterization of the GSY1 promoter also revealed a novel negative element. This element, N1, can repress expression driven by either an STRE or a heterologous element, the UAS of CYC1. Repression by N1 is dependent on the number of these elements that are present, but is independent of their orientation. N1 repressed expression when placed either upstream or downstream of the UAS, although the latter position is more effective. Gel shift analysis detected a factor that appears to bind to the N1 element. The complexity of the GSY1 promoter, which includes two STREs and three distinct negative elements, was surprising. This complexity may allow GSY1 to respond to a wide range of environmental stresses. GSY1 is one of the two genes encoding glycogen synthase in Saccharomyces cerevisiae. Both the GSY1 message and the protein levels increased as cells approached stationary phase. A combination of deletion analysis and site-directed mutagenesis revealed a complex promoter containing multiple positive and negative regulatory elements. Expression of GSY1 was dependent upon the presence of a TATA box and two stress response elements (STREs). Expression was repressed by Mig1, which mediates responses to glucose, and Rox1, which mediates responses to oxygen. Characterization of the GSY1 promoter also revealed a novel negative element. This element, N1, can repress expression driven by either an STRE or a heterologous element, the UAS of CYC1. Repression by N1 is dependent on the number of these elements that are present, but is independent of their orientation. N1 repressed expression when placed either upstream or downstream of the UAS, although the latter position is more effective. Gel shift analysis detected a factor that appears to bind to the N1 element. The complexity of the GSY1 promoter, which includes two STREs and three distinct negative elements, was surprising. This complexity may allow GSY1 to respond to a wide range of environmental stresses. The yeast Saccharomyces cerevisiae is exposed to a wide variety of environmental stressors, such as growth into stationary phase, heat shock, and osmotic shock. Cells respond to these stressors by modifying many metabolic processes, presumably making cells more resistant to these stressors. Among these changes are increased activities of the glycogen metabolic enzymes and the deposition of glycogen up to 23% of the dry weight of the cell (see below). Glycogen metabolism is highly conserved from yeast to mammals. The regulation of glycogen metabolism in S. cerevisiae closely parallels the more extensively studied counterparts in mammals (1Lillie S.H. Pringle J.R. J. Bacteriol. 1980; 143: 1384-1394Crossref PubMed Google Scholar, 2Roach P.J. Curr. Mol. Med. 2002; 2: 101-120Crossref PubMed Scopus (348) Google Scholar). Regulation is mediated primarily by effects on the activities of glycogen synthase and glycogen phosphorylase. These enzymes are regulated, in part, by protein phosphorylation. Cyclic AMP appears to play a central role in the regulation of these enzymes in S. cerevisiae, as it does in mammals, although the precise mechanisms remain to be identified. The parallels between glycogen metabolism in S. cerevisiae and mammals extends to the level of protein sequence. Glycogen synthase and glycogen phosphorylase from this yeast and mammals are 50 and 49% identical, respectively (3Farkas I. Hardy T.A. DePauli-Roach A.A. Roach P.J. J. Biol. Chem. 1990; 265: 20879-20886Abstract Full Text PDF PubMed Google Scholar, 4Farkas I. Hardy T.A. Goebl M.G. Roach P.J. J. Biol. Chem. 1991; 266: 15602-15607Abstract Full Text PDF PubMed Google Scholar, 5Huang K.P. Cabib E. J. Biol. Chem. 1974; 249: 3851-3857Abstract Full Text PDF PubMed Google Scholar, 6Hwang P.K. Fletterick R.J. Nature. 1986; 324: 80-84Crossref PubMed Scopus (62) Google Scholar). Glycogen metabolism is also regulated at the level of gene expression in S. cerevisiae. The protein levels of the enzymes involved in glycogen metabolism increase in parallel with glycogen accumulation as cells approach stationary phase or when nutrients are depleted (7Becker J.-U. J. Gen. Microbiol. 1982; 128: 447-454PubMed Google Scholar, 8Rothman-Denes L.B. Cabib E. Proc. Natl. Acad. Sci. U. S. A. 1970; 66: 967-974Crossref PubMed Scopus (83) Google Scholar). This increase in the level of glycogen metabolic enzyme activity appears to result, in part, from the regulation at the level of transcription. Northern blot analysis has shown that the levels of mRNA expressed from GPH1 (encoding glycogen phosphorylase), GLC3 (glycogen branching enzyme), GAC1 (glycogen-binding subunit of protein phosphatase 1), and GSY2 (glycogen synthase) increase as cells progress from log phase to stationary phase (9Hwang P.K. Tugendreich S. Fletterick R.J. Mol. Cell. Biol. 1989; 9: 1659-1666Crossref PubMed Scopus (70) Google Scholar, 10Rowen D.W. Meinke M. LaPorte D.C. Mol. Cell. Biol. 1992; 12: 22-29Crossref PubMed Scopus (43) Google Scholar, 11Francois J.M. Thompson-Jaeger S. Skroch U. Zellenka U. Spevak W. Tatchell K. EMBO J. 1992; 11: 87-96Crossref PubMed Scopus (126) Google Scholar, 12Ni H.T. LaPorte D.C. Mol. Microbiol. 1995; 16: 1197-1205Crossref PubMed Scopus (44) Google Scholar). The simultaneous increases in mRNA levels of the proteins involved in glycogen metabolism suggest that the expression of these genes may be coordinately regulated. Our long term goal is to understand how glycogen metabolism is regulated at the level of gene expression and how these regulatory processes are coordinated with post-translational control of the glycogen metabolic enzymes. The first step toward this goal is to characterize the promoters for these genes. In this paper, we report a characterization of the promoter of GSY1 (glycogen synthase). This is a surprisingly complex promoter that allows transcription to respond to a wide variety of cellular stressors. Materials—Restriction enzymes, other DNA modification enzymes and oligonucleotides linkers were purchased from Invitrogen or New England Biolabs. Radiochemicals were obtained from PerkinElmer Life Sciences and Amersham, Amplitaq polymerase for PCR from PerkinElmer Life Sciences, and sequencing reagents from U. S. Biochemical Corp. Oligonucleotide primers were products of National BioSciences, Inc. or Invitrogen. The frozen E-Z transformation kit was purchased from Zymo Research, Orange, CA. All other reagents were of the purest grades available. Isolation of the GSY1 Clone—GSY1, the gene encoding yeast glycogen synthase, was isolated as described by Meinke in 1993 (13Meinke, M. H. (1993) Regulation of Expression of a Gene for Glycogen Synthase in Saccharomyces cerevisiae. Ph.D. thesis, University of MinnesotaGoogle Scholar). Briefly, glycogen synthase was purified from strain S288C cells grown on YPD medium. The purified protein was reduced, carboxymethylated, and digested with trypsin. Two peptides were purified by reverse phase high performance liquid chromatography, their sequences were determined, and oligonucleotide probes were prepared. GSY1 was isolated following screening of a yeast genomic library in λ dash (Stratagene Cloning Systems) by plaque hybridization. The identities of the clones were confirmed by nucleotide sequence analysis and they were recloned as SalI fragments into plasmid vector pSEY18 (14Emr S.D. Vassarotti A. Garrett J. Geller B.L. Takeda M. Douglas M.G. J. Cell Biol. 1986; 102: 523-533Crossref PubMed Scopus (139) Google Scholar). Yeast Methods—Yeast strains were manipulated by standard methods unless indicated otherwise (15Sherman F. Fink G.R. Hicks J.B. Methods in Yeast Genetics. Cold Spring Harbor Laboratory, Cold Spring Harbor, NY1985Google Scholar). Transformation of linear DNA fragments was done by either the lithium acetate transformation method (16Schiestl R.H. Gietz R.D. Curr. Genet. 1989; 16: 339-346Crossref PubMed Scopus (1773) Google Scholar) or using the E-Z yeast transformation kit supplied by Zymo Research, Inc. Northern analysis was performed by standard procedures (17Ausubel F.M. Brent R. Kingston R.E. Moore D.D. Smith J.A. Seidman J.G. Struhl K. Current Protocols in Molecular Biology. John Wiley and Sons, New York1987Google Scholar). Constructs were transferred to the CAN1 locus of the chromosome, unless otherwise noted. Transfer was accomplished using the integrating plasmid pRL95 (12Ni H.T. LaPorte D.C. Mol. Microbiol. 1995; 16: 1197-1205Crossref PubMed Scopus (44) Google Scholar, 18Rowen, D. W. (1992) GLC3: The Gene Encoding the Glycogen Branching Enzyme of Saccharomyces cerevisiae. Ph.D thesis, University of MinnesotaGoogle Scholar). This vector includes two fragments of the CAN1 gene. Digestion with a restriction enzyme that cuts between these segments produces a linear fragment that will integrate into the CAN1 locus, replacing the resident sequences and producing a stable, single-copy integrant. Integrants were selected as uracil prototrophs (because of the URA3 gene carried by the vector) and then confirmed by resistance to canavanine plates and sensitivity to 5-fluoroorotic acid. Culture Conditions—Yeast cells were grown either in YPD (2% glucose, 2% peptone, and 1% yeast extract) or in SD media (2% glucose, 0.5% ammonium sulfate, and 0.17% yeast nitrogen base minus the amino acids) supplemented with the appropriate nutritional requirements (15Sherman F. Fink G.R. Hicks J.B. Methods in Yeast Genetics. Cold Spring Harbor Laboratory, Cold Spring Harbor, NY1985Google Scholar). 5-Fluoroorotic acid plates were prepared as described (19Boeke J.D. Trueheart J. Natsoulis G. Fink G.R. Methods Enzymol. 1987; 154: 164-175Crossref PubMed Scopus (1076) Google Scholar). SD + CAN plates contained SD supplemented with canavanine at a concentration of 60 μg/ml (20Sikorski R.S. Boeke J.D. Methods Enzymol. 1991; 194: 302-318Crossref PubMed Scopus (495) Google Scholar). GSY1 expression was routinely induced by growth into early stationary phase. Growth was monitored by light scattering at 600 nm. Cultures were grown at 30 °C and samples were taken at early log phase 600 between and or in early stationary phase heat were grown at °C to an A 600 of to was transferred into of the two was at °C and the other at The was The control and the samples were was by activity (see below). of the sequence of GSY1 was digested with at base to the were with fragment of DNA polymerase and linkers were The which includes the of was into plasmid D. Fink G.R. Gene 1987; PubMed Scopus Google Scholar). A gene from A. J. Methods Enzymol. PubMed Scopus Google Scholar) was then into the The plasmid a gene that includes the first of the GSY1 gene with the gene and has of GSY1 upstream This was into the to of and was for or in were using restriction mutagenesis of the was performed with the strain was for the A GSY1 upstream sequence from was into a The for mutagenesis in the of the to a restriction in the vector) and this screening for the which were then by sequencing with kit S. Biochemical The upstream sequence was then to the sequences in The plasmid was into the yeast chromosome, as described The of oligonucleotides in this can be in of oligonucleotides for this in a of to N1 the N1 element, of and were at °C for in containing and 50 The were then to to a of The stress response stress response of GSY1 and the sequences to were upstream of a N1 oligonucleotide was into restriction either downstream or upstream of the sequence. The were by of these into the CAN1 locus, these strains were grown in and activity was in early log phase and in stationary phase The is of for and The from plasmid Cell. Full Text PDF PubMed Scopus Google Scholar) was upstream of a N1 was then into restriction either downstream of the or upstream of the of the of these into the CAN1 locus, these strains were grown in to an early log phase and cells were for activity at an A 600 of analysis was performed with of a J. J. Bacteriol. 1992; PubMed Google Scholar). was obtained at the indicated from strain either on a plasmid or the control vector oligonucleotide to the sequence to to the of was as a and was using of was with and the was to with This was to to 50 °C and of and and of reverse were to the The was then to at °C for The products were on an The of GSY1 were by with a that was the products of The sequencing were carried using the as that for cell samples were by and on dry the the was and in and 50 and Cells were by in the presence of and the protein was obtained by The was then at 30 °C with and was at in Molecular Genetics. Cold Spring Harbor Laboratory, Cold Spring Harbor, Scholar). concentration was by the method of R.J. J. Biol. Chem. Full Text PDF PubMed Google using as the activities are for the protein concentration in the cell activities are the of three or more independent two independent were for In was of the strain was grown in of YPD to an A 600 of Cells were by in ammonium sulfate, and μg/ml and then in of were transferred to containing of and frozen in a dry the they were in the for of was and the samples were to and cell The was isolated and by at for at was by ammonium to The was by and in of μg/ml and were of the for The were and were and at The N1 oligonucleotide was using and The was then a to The were carried in a of containing of DNA 1% and of and of A contained or of and 30 of yeast at for 30 the samples were on a containing and for at were performed using N1, of the N1 oligonucleotide in which in the N1 and The N1 was and the Regulation of GSY1 Expression by Growth activity of yeast glycogen synthase increases as approach stationary phase L.B. Cabib E. Proc. Natl. Acad. Sci. U. S. A. 1970; 66: 967-974Crossref PubMed Scopus (83) Google Scholar). This increase in activity be of a in the of the glycogen synthase protein to an increase in the protein level as cells approach stationary phase. this a gene containing of upstream GSY1 sequence was and into the of strain The activity of this was in the early log phase of an increase be as early as the phase and a increase was upon into stationary phase of growth otherwise growth to stationary phase was as the standard method for of GSY1 expression in this The level of the GSY1 mRNA also increased as the stationary phase that at the level of transcription. of the of GSY1 was by analysis A was upstream of the of the with multiple or for other genes in yeast S. Proc. Natl. Acad. Sci. U. S. A. PubMed Scopus Google Scholar, S. Fink G.R. Proc. Natl. Acad. Sci. U. S. A. PubMed Scopus Google Scholar). All in this are to the of GSY1 transcription. The sequence which is an with the for TATA elements, was at to the of this TATA sequence was to was a in the expression of GSY1 in log and stationary phase cells the was with that of the appears that this sequence is a TATA and that this is involved with regulation in response to growth GSY1 Expression and STREs and and we that GPH1 and which glycogen phosphorylase and an of glycogen synthase in S. cerevisiae, STRE to expression in response to stationary phase and heat (12Ni H.T. LaPorte D.C. Mol. Microbiol. 1995; 16: 1197-1205Crossref PubMed Scopus (44) Google Scholar, I. LaPorte D.C. PubMed Scopus Google Scholar). that to the for STREs were also in the promoter of at and These elements were as and respectively of either or or the of upstream sequences in a in expression of GSY1 in log and stationary phase these elements to be and are for expression of These elements may the of the activities with either was that when were The factor in yeast is the of the gene. a of was also shown to be of the STRE sequence G. A. H. F. EMBO J. PubMed Scopus Google Scholar, K. Proc. Natl. Acad. Sci. U. S. A. PubMed Scopus Google Scholar). of and genes activity with the that they on GSY1 the STRE of a number of genes in response to a variety of stressors in to stationary phase G. G. H. EMBO J. 12: PubMed Scopus Google Scholar). a of the of and we the response of GSY1 to heat shock. a from to °C a of GSY1 promoter activity an following the shift of either this response and the to respond at with the of growth into stationary phase, and to GSY1 a deletion analysis of the GSY1 promoter, we a between and that to a negative This deletion in a increase in promoter activity in log and stationary phase of of this a sequence that the for a protein that is induced in response to R.S. Microbiol. 1992; PubMed Google Scholar). the role of this in GSY1 promoter we this sequence to in the of the gene increased expression of a in log phase and stationary cells with the that the of the in a increased In of this in a of the gene a on expression of the This from the presence of a in the GSY1 promoter, we other sequence that the The of the gene may a of a on cellular although that to be The gene is to be by R.S. Mol. Cell. Biol. PubMed Scopus Google Scholar). GSY1 repressed by Rox1, then a be to GSY1 expression of a in this by the genes into strains that carried or of a gene for Mol. Cell. Biol. 1992; 12: PubMed Scopus Google Scholar). of with the The in the a was by the of and the were appears that GSY1 and may an of be that the strains in A and of levels of This is by the that these strains GSY1 of the GSY1 promoter sequence revealed a at This sequence the of M. H. Mol. Cell. Biol. PubMed Scopus Google Scholar, H. EMBO J. 1990; 9: PubMed Scopus Google a protein that in of this a increase in expression A increase was when the gene was These effects were of the gene and the the as either These that GSY1 is repressed by when grown on The that appears to repress GSY1 expression that this gene respond to as a with this the levels of GSY1 protein were on with growth on or of a in the GSY1 the GSY1 promoter revealed a negative element. between and a increase in expression analysis indicated that the negative at in part, between to and effects up to were when the was the of this negative by mutagenesis or of the from to on that the negative this A revealed sequence in the yeast that this that this may be to this negative as N1 was to repress expression from an promoter, we an N1 oligonucleotide that the sequence from to This oligonucleotide was downstream of in the promoter the TATA and regulatory of This high promoter activity in the log phase in the of negative elements and an in levels as cells stationary phase. A of N1 was to repress expression from in log and stationary more the activity was induced when the cells stationary phase, that These were obtained with N1 in either orientation. Two or more of N1 expression In when the oligonucleotide which in base of the of N1, was placed downstream of it a in expression and also the when these cells stationary phase the appears to be to the novel N1 sequence. upstream of a of N1 in either a in In to downstream of a upstream of N1 when of N1 were upstream of a of was and the upon stationary phase was the effects of N1 on a heterologous promoter, the UAS from CYC1. The of N1 on this UAS was to on although the was N1 repressed expression upstream and downstream of this UAS, although the latter was more effective. Repression with the in Two of the were more Gel shift were carried to for N1 activities in The N1 oligonucleotide was and with the yeast and three were of N1 oligonucleotide a in the of the in the presence of of the the in of the oligonucleotide was This be the activity was in of the In the oligonucleotide and the DNA on the of this Two of the were to by and A the of the and may be to it appears that the a N1 This activity may the activity of N1, although this to be Glycogen synthase is a control for glycogen metabolism in S. cerevisiae, as it is in mammals (1Lillie S.H. Pringle J.R. J. Bacteriol. 1980; 143: 1384-1394Crossref PubMed Google Scholar). In to post-translational control mediated by protein and mechanisms Roach P.J. J. Biol. Chem. Full Text Full Text PDF PubMed Google glycogen synthase in yeast is regulated at the level of gene The promoter of one of the two genes encoding glycogen synthase, is surprisingly complex Expression is dependent on two for the and were identified. GSY1 may be the gene promoter for of these A novel negative was also STREs in a number of stress genes in S. cerevisiae. These elements respond to a variety of stressors, such as stationary phase, heat shock, and osmotic H. 1995; PubMed Scopus Google Scholar). and proteins shown to be for the STREs in S. cerevisiae G. A. H. F. EMBO J. PubMed Scopus Google Scholar, K. Proc. Natl. Acad. Sci. U. S. A. PubMed Scopus Google Scholar, H. G. F. E. M. PubMed Scopus Google Scholar). GSY1 has two of the STRE that with of GSY1 expression these STREs was in a role for these genes in the stress response of is a protein that was first of CYC1. Expression of to the levels of and of or levels are reduced, expression of genes. also appears to control GSY1 of either the gene or of the the GSY1 promoter increased the expression of a The of to repress GSY1 that expression of this gene respond to and R.S. Microbiol. 1992; PubMed Google Scholar, R.S. Mol. Cell. Biol. PubMed Scopus Google Scholar). with this growth and D. and of gene for increased expression these be with growth and the to be to GSY1 expression may respond to levels the has shown to play a central role in of a variety of genes. of the gene or the the GSY1 promoter increased expression of a a role for this protein in the control of growth on a in expression with growth on or which also that GSY1 to as with the response to the be with of the complexity of the effects by these growth has shown to be by the yeast of the Google Scholar, M. Curr. Microbiol. 2: PubMed Scopus Google Scholar, J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar, S. M. Mol. Cell. Biol. PubMed Scopus Google Scholar). AMP levels a of in the the of and of the genes Curr. Biol. Full Text Full Text PDF PubMed Scopus Google Scholar). glycogen levels S. J. Tatchell K. Genetics. 1991; PubMed Google with a role for in the glycogen synthase genes. The GSY1 promoter also a novel negative element, which we to as This transcription when either upstream or downstream of a UAS, although the latter position is more effective. N1 is in either and is more when multiple are is to at the UAS of as The role of N1 in GSY1 expression has to be it is that this can from a of an STRE when it does repress are of negative in yeast with a that N1 is a element. is the promoter of GSY1 Glycogen in response to a wide variety of stressors, such as into stationary phase, heat shock, and osmotic J. Microbiol. PubMed Scopus Google Scholar, M. J. 143: PubMed Scopus Google Scholar, A. J. PubMed Scopus Google Scholar). accumulation is it increases the of glycogen that is to the cell the metabolic The response of GSY1 to that appears to be mediated by is also to be glycogen can be The complexity of the GSY1 promoter may that glycogen synthase is induced in response to a wide variety of stressors. Yeast glycogen synthase is by two GSY1 and Both promoters STRE that to the of the gene upon into stationary phase. The GSY1 promoter also includes a number of negative elements, such elements in the GSY2 promoter (12Ni H.T. LaPorte D.C. Mol. Microbiol. 1995; 16: 1197-1205Crossref PubMed Scopus (44) Google Scholar). GSY2 has to glycogen synthase at a level does GSY1 and is to be the to glycogen growth the presence of negative elements in but that the of GSY1 may the of glycogen synthase that from these elements. These negative elements a range of expression levels and the to respond to a of metabolic be obtained with STRE R. for strains and H. for strains and for strains and and for plasmid