Glucose-mediated Phosphorylation Converts the Transcription Factor Rgt1 from a Repressor to an Activator
2003; Elsevier BV; Volume: 278; Issue: 12 Linguagem: Inglês
10.1074/jbc.m212802200
ISSN1083-351X
AutoresAmber L. Mosley, Jaganathan Lakshmanan, Bishwa K. Aryal, Sabire Özcan,
Tópico(s)Biofuel production and bioconversion
ResumoGlucose, the most abundant carbon and energy source, regulates the expression of genes required for its own efficient metabolism. In the yeast Saccharomyces cerevisiae, glucose induces the expression of the hexose transporter (HXT) genes by modulating the activity of the transcription factor Rgt1 that functions as a repressor when glucose is absent. However, in the presence of high concentrations of glucose, Rgt1 is converted from a repressor to an activator and is required for maximal induction of HXT1 gene expression. We report that Rgt1 binds to the HXT1 promoter only in the absence of glucose, suggesting that Rgt1 increases HXT1 gene expression at high levels of glucose by an indirect mechanism. It is likely that Rgt1 stimulates the expression of an activator of theHXT1 gene at high concentrations of glucose. In addition, we demonstrate that Rgt1 becomes hyperphosphorylated in response to high glucose levels and that this phosphorylation event is required for Rgt1 to activate transcription. Furthermore, Rgt1 lacks the glucose-mediated phosphorylation in the snf3 rgt2 andgrr1 mutants, which are defective in glucose induction ofHXT gene expression. In these mutants, Rgt1 behaves as a constitutive repressor independent of the carbon source. We conclude that phosphorylation of Rgt1 in response to glucose is required to abolish the Rgt1-mediated repression of the HXT genes and to convert Rgt1 from a transcriptional repressor to an activator. Glucose, the most abundant carbon and energy source, regulates the expression of genes required for its own efficient metabolism. In the yeast Saccharomyces cerevisiae, glucose induces the expression of the hexose transporter (HXT) genes by modulating the activity of the transcription factor Rgt1 that functions as a repressor when glucose is absent. However, in the presence of high concentrations of glucose, Rgt1 is converted from a repressor to an activator and is required for maximal induction of HXT1 gene expression. We report that Rgt1 binds to the HXT1 promoter only in the absence of glucose, suggesting that Rgt1 increases HXT1 gene expression at high levels of glucose by an indirect mechanism. It is likely that Rgt1 stimulates the expression of an activator of theHXT1 gene at high concentrations of glucose. In addition, we demonstrate that Rgt1 becomes hyperphosphorylated in response to high glucose levels and that this phosphorylation event is required for Rgt1 to activate transcription. Furthermore, Rgt1 lacks the glucose-mediated phosphorylation in the snf3 rgt2 andgrr1 mutants, which are defective in glucose induction ofHXT gene expression. In these mutants, Rgt1 behaves as a constitutive repressor independent of the carbon source. We conclude that phosphorylation of Rgt1 in response to glucose is required to abolish the Rgt1-mediated repression of the HXT genes and to convert Rgt1 from a transcriptional repressor to an activator. hemagglutinin immunoprecipitation chromatin immunoprecipitation The yeast Saccharomyes cerevisiae uses glucose as its preferred carbon and energy source. Glucose not only represses the expression of genes that are required for the metabolism of alternate sugars but also induces the transcription of genes that are essential for its own efficient utilization (1Gancedo J.M. Microbiol. Mol. Biol. Rev. 1998; 62: 334-361Crossref PubMed Google Scholar, 2Carlson M. Curr. Opin. Genet. Dev. 1998; 8: 560-564Crossref PubMed Scopus (72) Google Scholar, 3Johnston M. Trends Genet. 1999; 15: 29-33Abstract Full Text Full Text PDF PubMed Scopus (318) Google Scholar). Among the genes that are induced by glucose are the members of the HXT gene family, which encode glucose transporters. Glucose induces the expression of the HXT1–HXT4 genes by 10–300-fold (4Özcan S. Johnston M. Microbiol. Mol. Biol. Rev. 1999; 63: 554-569Crossref PubMed Google Scholar, 5Özcan S. Johnston M. Mol. Cell. Biol. 1995; 15: 1564-1572Crossref PubMed Google Scholar).Several components of the glucose induction pathway required forHXT gene expression have been identified, including the glucose sensors Snf3 and Rgt2, that are responsible for sensing extracellular glucose and generating the intracellular signal (6Özcan S. Dover J. Rosenwald A.G. Wölfl S. Johnston M. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 12428-12432Crossref PubMed Scopus (341) Google Scholar, 7Özcan S. Dover J. Johnston M. EMBO J. 1998; 17: 2566-2573Crossref PubMed Scopus (285) Google Scholar). A strain mutated for both sensors (snf3 rgt2 double mutant) is completely defective in glucose induction of the HXT gene expression (7Özcan S. Dover J. Johnston M. EMBO J. 1998; 17: 2566-2573Crossref PubMed Scopus (285) Google Scholar). Another component that is absolutely essential for glucose induction of the HXT gene expression is the ubiquitin ligase Grr1 (5Özcan S. Johnston M. Mol. Cell. Biol. 1995; 15: 1564-1572Crossref PubMed Google Scholar, 8Li F.N. Johnston M. EMBO J. 1997; 16: 5629-5638Crossref PubMed Scopus (182) Google Scholar). Two homologous proteins, Std1 and Mth1, have been shown to negatively regulate HXT gene expression and to interact with the carboxyl-terminal tails of the Snf3 and Rgt2 sensors (9Schmidt M.C. McCartney R.R. Zhang X. Tillman T.S. Solimeo H. Wolfl S. Almonte C. Watkins S.C. Mol. Cell. Biol. 1999; 19: 4561-4571Crossref PubMed Scopus (129) Google Scholar, 10Schulte F. Wieczorke R. Hollenberg C.P. Boles E. J. Bacteriol. 2000; 182: 540-542Crossref PubMed Scopus (43) Google Scholar, 11Lafuente M. Gancedo J.C. Jauniaux J.-C. Gancedo J.M. Mol. Microbiol. 2000; 35: 161-172Crossref PubMed Scopus (86) Google Scholar). Repression of HXT gene expression in the absence of glucose is abolished in a std1 mth1 double mutant (9Schmidt M.C. McCartney R.R. Zhang X. Tillman T.S. Solimeo H. Wolfl S. Almonte C. Watkins S.C. Mol. Cell. Biol. 1999; 19: 4561-4571Crossref PubMed Scopus (129) Google Scholar, 11Lafuente M. Gancedo J.C. Jauniaux J.-C. Gancedo J.M. Mol. Microbiol. 2000; 35: 161-172Crossref PubMed Scopus (86) Google Scholar).The target of the glucose induction signal is the Cys6DNA-binding protein Rgt1, which belongs to the family of the Gal4 transcription factors (12Özcan S. Leong T. Johnston M. Mol. Cell. Biol. 1996; 16: 6419-6426Crossref PubMed Scopus (134) Google Scholar). In the absence of glucose, Rgt1 repressesHXT gene expression, whereas at high concentrations of glucose Rgt1 is required for maximal activation of HXT1 gene expression. Repression of transcription by Rgt1 in the absence of glucose requires the general repressor complex, Ssn6 and Tup1. Activation of transcription by Rgt1 in response to high concentrations of glucose requires the glucose sensors Snf3 and Rgt2 and the ubiquitin ligase Grr1 (12Özcan S. Leong T. Johnston M. Mol. Cell. Biol. 1996; 16: 6419-6426Crossref PubMed Scopus (134) Google Scholar).It was previously shown that Rgt1 is required for both repression and activation of HXT1 gene expression and binds to theHXT1 promoter in vitro by gel shift assays (12Özcan S. Leong T. Johnston M. Mol. Cell. Biol. 1996; 16: 6419-6426Crossref PubMed Scopus (134) Google Scholar). We report that the requirement of Rgt1 for activation ofHXT1 gene expression in response to high concentrations of glucose is mediated by an indirect mechanism, because Rgt1 is unable to bind to the HXT1 gene promoter in vivo when high levels of glucose are present. Previous data indicated that the transcription of Rgt1 is not regulated by glucose, suggesting the idea that the transcriptional activity of Rgt1 is regulated post-translationally (12Özcan S. Leong T. Johnston M. Mol. Cell. Biol. 1996; 16: 6419-6426Crossref PubMed Scopus (134) Google Scholar). We demonstrate that Rgt1 is hyperphosphorylated in response to high concentrations of glucose and that the lack of Rgt1 phosphorylation abolishes its ability to activate transcription.DISCUSSIONIt has been previously shown that Rgt1 is the target of the glucose induction pathway that leads to up-regulation of HXTgene expression (5Özcan S. Johnston M. Mol. Cell. Biol. 1995; 15: 1564-1572Crossref PubMed Google Scholar). Furthermore, it was demonstrated that Rgt1 is a bifunctional transcription factor that is converted from a transcriptional repressor to an activator in response to high concentrations of glucose (12Özcan S. Leong T. Johnston M. Mol. Cell. Biol. 1996; 16: 6419-6426Crossref PubMed Scopus (134) Google Scholar). In this publication, we have addressed the question of whether glucose regulates the transcriptional activity of Rgt1 by mediating changes in Rgt1 subcellular localization, DNA binding, or post-translational modification. Several transcription factors have been shown to change their subcellular localization in response to external stimuli in yeast, including Mig1 and Pho4 (19DeVit M.J.D. Waddle J.A. Johnston M. Mol. Biol. Cell. 1997; 8: 1603-1618Crossref PubMed Scopus (282) Google Scholar,23Komeili A. O'Shea E.K. Science. 1999; 284: 977-980Crossref PubMed Scopus (245) Google Scholar). Mig1 is phosphorylated by the Snf1 kinase in the absence of glucose or presence of low levels of glucose and is trapped in the cytoplasm. High concentrations of glucose cause dephosphorylation of Mig1 and translocation into the nucleus (19DeVit M.J.D. Waddle J.A. Johnston M. Mol. Biol. Cell. 1997; 8: 1603-1618Crossref PubMed Scopus (282) Google Scholar, 20Treitel M.A. Kuchin S. Carlson M. Mol. Cell. Biol. 1998; 18: 6273-6280Crossref PubMed Scopus (260) Google Scholar). The subcellular localization of Pho4 is regulated in response to phosphate. At high concentrations of phosphate, Pho4 is phosphorylated and exported into the cytoplasm, whereas starvation for phosphate causes dephosphorylation of Pho4 and translocation into the nucleus (23Komeili A. O'Shea E.K. Science. 1999; 284: 977-980Crossref PubMed Scopus (245) Google Scholar). Our data obtained using indirect immunofluorescence microscopy suggest that the subcellular localization of Rgt1 does not change in response to glucose.We also tested the possibility that glucose may regulate the DNA binding affinity of Rgt1 utilizing the ChIP assay. As expected we did not see any significant binding of Rgt1 in vivo to theHXT2 and HXT3 promoters in cells grown in presence of high concentrations of glucose. Rgt1 binding to theHXT2 and HXT3 gene promoters was observed only in the absence of glucose, consistent with its role as a transcriptional repressor of the HXT2 and HXT3 genes when glucose is absent.Several lines of evidence indicate that Rgt1 regulates both repression and activation of HXT1 gene expression. First, Rgt1 is able to bind to a 280-bp promoter region of the HXT1 genein vitro that is sufficient for glucose induction of this gene (12Özcan S. Leong T. Johnston M. Mol. Cell. Biol. 1996; 16: 6419-6426Crossref PubMed Scopus (134) Google Scholar).2 Second, deletion of RGT1 causes a 10-fold increase in HXT1 expression when glucose is absent but a 5–6-fold decrease in HXT1 gene expression at high concentrations of glucose. Third, a lexA-Rgt1 fusion protein functions as a repressor of transcription in the absence of glucose and as an activator of transcription when glucose is abundant (12Özcan S. Leong T. Johnston M. Mol. Cell. Biol. 1996; 16: 6419-6426Crossref PubMed Scopus (134) Google Scholar). These data suggested that Rgt1 might directly regulate HXT1 gene expression in the absence and presence of high concentrations of glucose. However, ChIP analysis of Rgt1 binding in vivoindicates that the positive regulatory effect of Rgt1 onHXT1 gene expression is mediated by an indirect mechanism, because Rgt1 is unable to bind to the HXT1 promoter in response to glucose. It is likely that Rgt1 is required for expression of a transcriptional activator of the HXT1 gene at high concentrations of glucose (Fig. 7).We have consistently observed a change in Rgt1 molecular weight in response to glucose, suggesting that glucose regulates the transcriptional activity of Rgt1 by a post-translational modification event. Treatment of extracts of glucose-grown cells with λ phosphatase indicated that Rgt1 is phosphorylated in response to glucose. Because the molecular mass of the lexA-Rgt1 fusion construct is about 140 kDa, the observed shift in Rgt1 mobility at high concentrations of glucose is likely due to hyperphosphorylation or phosphorylation at multiple sites. The activity of several eukaryotic transcription factors is regulated by reversible phosphorylation in response to changes in the cellular environment. Multisite phosphorylation in particular has been shown to provide a dynamic and precise tuning of the transactivating potential of transcription factors rather than being a static on/off switch. Multisite phosphorylation of transcription factors such as heat shock factor 1 and p53 enables the integration of several different signals (24Holmberg C.I. Tran S.E.F. Eriksson J.E. Sistonen L. Trends Biochem. Sci. 2002; 27: 619-627Abstract Full Text Full Text PDF PubMed Scopus (239) Google Scholar). Multisite phosphorylation of Rgt1 could have several functions such as the disruption of Rgt1 interaction with a repressor complex in the absence of glucose (Fig. 7) and conversion of Rgt1 from a transcriptional repressor to an activator via unmasking of a specific activation domain.Based on several lines of evidence, we propose that Rgt1 functions as a transcriptional repressor of the HXT genes in the absence of glucose, in a complex with the Ssn6-Tup1 and Mth1-Std1 repressor proteins (Fig. 7) (12Özcan S. Leong T. Johnston M. Mol. Cell. Biol. 1996; 16: 6419-6426Crossref PubMed Scopus (134) Google Scholar). 3J. Lakshmanan, A. L. Mosley, and S.Özcan, submitted for publication. When glucose is abundant, Rgt1 becomes hyperphosphorylated and dissociates from the repressor complex. Hyperphosphorylation also converts Rgt1 from a transcriptional repressor to an activator (Fig. 7). Consistent with this idea, deletion of the region from amino acids 75 to 395 abolishes the ability of Rgt1 to both activate transcription and become hyperphosphorylated. Furthermore, the grr1 and snf3 rgt2 mutant strains, where Rgt1 functions always as a transcriptional repressor independent of glucose, lack the glucose-induced modification of Rgt1. The presented data also suggest that Rgt1 is not directly involved in the activation of theHXT1 gene expression but rather stimulates the expression of a transcription factor that is required for maximal expression of theHXT1 gene when glucose is abundant (Fig. 7).The region of Rgt1 required for its glucose-mediated phosphorylation (amino acids 75–395) has several potential phosphorylation sites. Our preliminary data indicate that nuclear localization of Rgt1 is required for the glucose-induced modification, suggesting the idea that the kinase(s) that phosphorylate Rgt1 in response to glucose is nuclear. We are currently testing whether any of the known nuclear kinases are involved in phosphorylation of Rgt1.The transcription factor Ume6 belongs to the same family of DNA-binding proteins as Rgt1 and is also a repressor and an activator of gene expression (26Bowdish K.S. Yuan H.E. Mitchell A.P. Mol. Cell. Biol. 1995; 15: 2955-2961Crossref PubMed Scopus (89) Google Scholar, 27Rubin-Bejerano I. Mandel S. Robzyk K. Kassir Y. Mol. Cell. Biol. 1996; 16: 2518-2526Crossref PubMed Scopus (119) Google Scholar, 28Steber C.M. Esposito R.E. Proc. Natl. Acad. Sci. U. S. A. 1995; 92: 12490-12494Crossref PubMed Scopus (66) Google Scholar). Ume6 in a complex with Sin3 and Rpd3 functions as a repressor; however, its interaction with Ime1 during meiosis converts Ume6 to an activator of early genes (29Washburn B.K. Esposito R.E. Mol. Cell. Biol. 2001; 21: 2057-2069291Crossref PubMed Scopus (75) Google Scholar). The co-repressor Sin3 interacts with the deacetylase Rpd3 and causes repression by deacetylation of histones (30Vidal M. Gaber R.F. Mol. Cell. Biol. 1991; 11: 6317-6327Crossref PubMed Scopus (261) Google Scholar, 31Vidal M. Strich R. Esposito R.E. Gaber R.F. Mol. Cell. Biol. 1991; 11: 6306-6316Crossref PubMed Scopus (142) Google Scholar, 32Kasten M.M. Dorland S. Stillman D.J. Mol. Cell. Biol. 1997; 17: 4852-4858Crossref PubMed Scopus (114) Google Scholar). Interaction of Ume6 with Ime1 is required for the phosphorylation of Ume6 at the amino terminus by the Rim11 and Mck1 kinases (33Xiao Y. Mitchell A.P. Mol. Cell. Biol. 2000; 20: 5447-5453Crossref PubMed Scopus (45) Google Scholar). Because Ime1 functions as a transcriptional activator when fused to a DNA-binding domain, it has been proposed that Ime1 provides the activation domain for Ume6 (22Mandel S. Robzyk K. Kassir Y. Dev. Genet. 1994; 15: 139-147Crossref PubMed Scopus (40) Google Scholar).Like with Ume6, phosphorylation of Rgt1 in response to high concentrations of glucose is important for Rgt1 to function as a transcriptional activator. However, it is not known whether phosphorylation facilitates Rgt1 to interact with an activator like Ime1. It is likely that repression by Rgt1 also involves a co-repressor complex like the Sin3 and Rpd3 that mediates deacetylation of histones. Indeed we have preliminary data indicating that glucose induction ofHXT genes involves changes in histone acetylation levels.2 Experiments are under way to test whether the observed changes in histone acetylation at the HXT gene promoters are mediated by Rgt1. In addition, being an activator itself, Ume6 also induces the expression of another transcription factor, Ndt80, that activates middle genes (25Hepworth S.R. Friesen H. Segall J. Mol. Cell. Biol. 1998; 18: 5750-5761Crossref PubMed Google Scholar). Similar to Ume6, Rgt1 is also likely to stimulate the expression of a transcriptional activator that is required for maximal expression of the HXT1 gene in response to high concentrations of glucose (Fig. 7). The yeast Saccharomyes cerevisiae uses glucose as its preferred carbon and energy source. Glucose not only represses the expression of genes that are required for the metabolism of alternate sugars but also induces the transcription of genes that are essential for its own efficient utilization (1Gancedo J.M. Microbiol. Mol. Biol. Rev. 1998; 62: 334-361Crossref PubMed Google Scholar, 2Carlson M. Curr. Opin. Genet. Dev. 1998; 8: 560-564Crossref PubMed Scopus (72) Google Scholar, 3Johnston M. Trends Genet. 1999; 15: 29-33Abstract Full Text Full Text PDF PubMed Scopus (318) Google Scholar). Among the genes that are induced by glucose are the members of the HXT gene family, which encode glucose transporters. Glucose induces the expression of the HXT1–HXT4 genes by 10–300-fold (4Özcan S. Johnston M. Microbiol. Mol. Biol. Rev. 1999; 63: 554-569Crossref PubMed Google Scholar, 5Özcan S. Johnston M. Mol. Cell. Biol. 1995; 15: 1564-1572Crossref PubMed Google Scholar). Several components of the glucose induction pathway required forHXT gene expression have been identified, including the glucose sensors Snf3 and Rgt2, that are responsible for sensing extracellular glucose and generating the intracellular signal (6Özcan S. Dover J. Rosenwald A.G. Wölfl S. Johnston M. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 12428-12432Crossref PubMed Scopus (341) Google Scholar, 7Özcan S. Dover J. Johnston M. EMBO J. 1998; 17: 2566-2573Crossref PubMed Scopus (285) Google Scholar). A strain mutated for both sensors (snf3 rgt2 double mutant) is completely defective in glucose induction of the HXT gene expression (7Özcan S. Dover J. Johnston M. EMBO J. 1998; 17: 2566-2573Crossref PubMed Scopus (285) Google Scholar). Another component that is absolutely essential for glucose induction of the HXT gene expression is the ubiquitin ligase Grr1 (5Özcan S. Johnston M. Mol. Cell. Biol. 1995; 15: 1564-1572Crossref PubMed Google Scholar, 8Li F.N. Johnston M. EMBO J. 1997; 16: 5629-5638Crossref PubMed Scopus (182) Google Scholar). Two homologous proteins, Std1 and Mth1, have been shown to negatively regulate HXT gene expression and to interact with the carboxyl-terminal tails of the Snf3 and Rgt2 sensors (9Schmidt M.C. McCartney R.R. Zhang X. Tillman T.S. Solimeo H. Wolfl S. Almonte C. Watkins S.C. Mol. Cell. Biol. 1999; 19: 4561-4571Crossref PubMed Scopus (129) Google Scholar, 10Schulte F. Wieczorke R. Hollenberg C.P. Boles E. J. Bacteriol. 2000; 182: 540-542Crossref PubMed Scopus (43) Google Scholar, 11Lafuente M. Gancedo J.C. Jauniaux J.-C. Gancedo J.M. Mol. Microbiol. 2000; 35: 161-172Crossref PubMed Scopus (86) Google Scholar). Repression of HXT gene expression in the absence of glucose is abolished in a std1 mth1 double mutant (9Schmidt M.C. McCartney R.R. Zhang X. Tillman T.S. Solimeo H. Wolfl S. Almonte C. Watkins S.C. Mol. Cell. Biol. 1999; 19: 4561-4571Crossref PubMed Scopus (129) Google Scholar, 11Lafuente M. Gancedo J.C. Jauniaux J.-C. Gancedo J.M. Mol. Microbiol. 2000; 35: 161-172Crossref PubMed Scopus (86) Google Scholar). The target of the glucose induction signal is the Cys6DNA-binding protein Rgt1, which belongs to the family of the Gal4 transcription factors (12Özcan S. Leong T. Johnston M. Mol. Cell. Biol. 1996; 16: 6419-6426Crossref PubMed Scopus (134) Google Scholar). In the absence of glucose, Rgt1 repressesHXT gene expression, whereas at high concentrations of glucose Rgt1 is required for maximal activation of HXT1 gene expression. Repression of transcription by Rgt1 in the absence of glucose requires the general repressor complex, Ssn6 and Tup1. Activation of transcription by Rgt1 in response to high concentrations of glucose requires the glucose sensors Snf3 and Rgt2 and the ubiquitin ligase Grr1 (12Özcan S. Leong T. Johnston M. Mol. Cell. Biol. 1996; 16: 6419-6426Crossref PubMed Scopus (134) Google Scholar). It was previously shown that Rgt1 is required for both repression and activation of HXT1 gene expression and binds to theHXT1 promoter in vitro by gel shift assays (12Özcan S. Leong T. Johnston M. Mol. Cell. Biol. 1996; 16: 6419-6426Crossref PubMed Scopus (134) Google Scholar). We report that the requirement of Rgt1 for activation ofHXT1 gene expression in response to high concentrations of glucose is mediated by an indirect mechanism, because Rgt1 is unable to bind to the HXT1 gene promoter in vivo when high levels of glucose are present. Previous data indicated that the transcription of Rgt1 is not regulated by glucose, suggesting the idea that the transcriptional activity of Rgt1 is regulated post-translationally (12Özcan S. Leong T. Johnston M. Mol. Cell. Biol. 1996; 16: 6419-6426Crossref PubMed Scopus (134) Google Scholar). We demonstrate that Rgt1 is hyperphosphorylated in response to high concentrations of glucose and that the lack of Rgt1 phosphorylation abolishes its ability to activate transcription. DISCUSSIONIt has been previously shown that Rgt1 is the target of the glucose induction pathway that leads to up-regulation of HXTgene expression (5Özcan S. Johnston M. Mol. Cell. Biol. 1995; 15: 1564-1572Crossref PubMed Google Scholar). Furthermore, it was demonstrated that Rgt1 is a bifunctional transcription factor that is converted from a transcriptional repressor to an activator in response to high concentrations of glucose (12Özcan S. Leong T. Johnston M. Mol. Cell. Biol. 1996; 16: 6419-6426Crossref PubMed Scopus (134) Google Scholar). In this publication, we have addressed the question of whether glucose regulates the transcriptional activity of Rgt1 by mediating changes in Rgt1 subcellular localization, DNA binding, or post-translational modification. Several transcription factors have been shown to change their subcellular localization in response to external stimuli in yeast, including Mig1 and Pho4 (19DeVit M.J.D. Waddle J.A. Johnston M. Mol. Biol. Cell. 1997; 8: 1603-1618Crossref PubMed Scopus (282) Google Scholar,23Komeili A. O'Shea E.K. Science. 1999; 284: 977-980Crossref PubMed Scopus (245) Google Scholar). Mig1 is phosphorylated by the Snf1 kinase in the absence of glucose or presence of low levels of glucose and is trapped in the cytoplasm. High concentrations of glucose cause dephosphorylation of Mig1 and translocation into the nucleus (19DeVit M.J.D. Waddle J.A. Johnston M. Mol. Biol. Cell. 1997; 8: 1603-1618Crossref PubMed Scopus (282) Google Scholar, 20Treitel M.A. Kuchin S. Carlson M. Mol. Cell. Biol. 1998; 18: 6273-6280Crossref PubMed Scopus (260) Google Scholar). The subcellular localization of Pho4 is regulated in response to phosphate. At high concentrations of phosphate, Pho4 is phosphorylated and exported into the cytoplasm, whereas starvation for phosphate causes dephosphorylation of Pho4 and translocation into the nucleus (23Komeili A. O'Shea E.K. Science. 1999; 284: 977-980Crossref PubMed Scopus (245) Google Scholar). Our data obtained using indirect immunofluorescence microscopy suggest that the subcellular localization of Rgt1 does not change in response to glucose.We also tested the possibility that glucose may regulate the DNA binding affinity of Rgt1 utilizing the ChIP assay. As expected we did not see any significant binding of Rgt1 in vivo to theHXT2 and HXT3 promoters in cells grown in presence of high concentrations of glucose. Rgt1 binding to theHXT2 and HXT3 gene promoters was observed only in the absence of glucose, consistent with its role as a transcriptional repressor of the HXT2 and HXT3 genes when glucose is absent.Several lines of evidence indicate that Rgt1 regulates both repression and activation of HXT1 gene expression. First, Rgt1 is able to bind to a 280-bp promoter region of the HXT1 genein vitro that is sufficient for glucose induction of this gene (12Özcan S. Leong T. Johnston M. Mol. Cell. Biol. 1996; 16: 6419-6426Crossref PubMed Scopus (134) Google Scholar).2 Second, deletion of RGT1 causes a 10-fold increase in HXT1 expression when glucose is absent but a 5–6-fold decrease in HXT1 gene expression at high concentrations of glucose. Third, a lexA-Rgt1 fusion protein functions as a repressor of transcription in the absence of glucose and as an activator of transcription when glucose is abundant (12Özcan S. Leong T. Johnston M. Mol. Cell. Biol. 1996; 16: 6419-6426Crossref PubMed Scopus (134) Google Scholar). These data suggested that Rgt1 might directly regulate HXT1 gene expression in the absence and presence of high concentrations of glucose. However, ChIP analysis of Rgt1 binding in vivoindicates that the positive regulatory effect of Rgt1 onHXT1 gene expression is mediated by an indirect mechanism, because Rgt1 is unable to bind to the HXT1 promoter in response to glucose. It is likely that Rgt1 is required for expression of a transcriptional activator of the HXT1 gene at high concentrations of glucose (Fig. 7).We have consistently observed a change in Rgt1 molecular weight in response to glucose, suggesting that glucose regulates the transcriptional activity of Rgt1 by a post-translational modification event. Treatment of extracts of glucose-grown cells with λ phosphatase indicated that Rgt1 is phosphorylated in response to glucose. Because the molecular mass of the lexA-Rgt1 fusion construct is about 140 kDa, the observed shift in Rgt1 mobility at high concentrations of glucose is likely due to hyperphosphorylation or phosphorylation at multiple sites. The activity of several eukaryotic transcription factors is regulated by reversible phosphorylation in response to changes in the cellular environment. Multisite phosphorylation in particular has been shown to provide a dynamic and precise tuning of the transactivating potential of transcription factors rather than being a static on/off switch. Multisite phosphorylation of transcription factors such as heat shock factor 1 and p53 enables the integration of several different signals (24Holmberg C.I. Tran S.E.F. Eriksson J.E. Sistonen L. Trends Biochem. Sci. 2002; 27: 619-627Abstract Full Text Full Text PDF PubMed Scopus (239) Google Scholar). Multisite phosphorylation of Rgt1 could have several functions such as the disruption of Rgt1 interaction with a repressor complex in the absence of glucose (Fig. 7) and conversion of Rgt1 from a transcriptional repressor to an activator via unmasking of a specific activation domain.Based on several lines of evidence, we propose that Rgt1 functions as a transcriptional repressor of the HXT genes in the absence of glucose, in a complex with the Ssn6-Tup1 and Mth1-Std1 repressor proteins (Fig. 7) (12Özcan S. Leong T. Johnston M. Mol. Cell. Biol. 1996; 16: 6419-6426Crossref PubMed Scopus (134) Google Scholar). 3J. Lakshmanan, A. L. Mosley, and S.Özcan, submitted for publication. When glucose is abundant, Rgt1 becomes hyperphosphorylated and dissociates from the repressor complex. Hyperphosphorylation also converts Rgt1 from a transcriptional repressor to an activator (Fig. 7). Consistent with this idea, deletion of the region from amino acids 75 to 395 abolishes the ability of Rgt1 to both activate transcription and become hyperphosphorylated. Furthermore, the grr1 and snf3 rgt2 mutant strains, where Rgt1 functions always as a transcriptional repressor independent of glucose, lack the glucose-induced modification of Rgt1. The presented data also suggest that Rgt1 is not directly involved in the activation of theHXT1 gene expression but rather stimulates the expression of a transcription factor that is required for maximal expression of theHXT1 gene when glucose is abundant (Fig. 7).The region of Rgt1 required for its glucose-mediated phosphorylation (amino acids 75–395) has several potential phosphorylation sites. Our preliminary data indicate that nuclear localization of Rgt1 is required for the glucose-induced modification, suggesting the idea that the kinase(s) that phosphorylate Rgt1 in response to glucose is nuclear. We are currently testing whether any of the known nuclear kinases are involved in phosphorylation of Rgt1.The transcription factor Ume6 belongs to the same family of DNA-binding proteins as Rgt1 and is also a repressor and an activator of gene expression (26Bowdish K.S. Yuan H.E. Mitchell A.P. Mol. Cell. Biol. 1995; 15: 2955-2961Crossref PubMed Scopus (89) Google Scholar, 27Rubin-Bejerano I. Mandel S. Robzyk K. Kassir Y. Mol. Cell. Biol. 1996; 16: 2518-2526Crossref PubMed Scopus (119) Google Scholar, 28Steber C.M. Esposito R.E. Proc. Natl. Acad. Sci. U. S. A. 1995; 92: 12490-12494Crossref PubMed Scopus (66) Google Scholar). Ume6 in a complex with Sin3 and Rpd3 functions as a repressor; however, its interaction with Ime1 during meiosis converts Ume6 to an activator of early genes (29Washburn B.K. Esposito R.E. Mol. Cell. Biol. 2001; 21: 2057-2069291Crossref PubMed Scopus (75) Google Scholar). The co-repressor Sin3 interacts with the deacetylase Rpd3 and causes repression by deacetylation of histones (30Vidal M. Gaber R.F. Mol. Cell. Biol. 1991; 11: 6317-6327Crossref PubMed Scopus (261) Google Scholar, 31Vidal M. Strich R. Esposito R.E. Gaber R.F. Mol. Cell. Biol. 1991; 11: 6306-6316Crossref PubMed Scopus (142) Google Scholar, 32Kasten M.M. Dorland S. Stillman D.J. Mol. Cell. Biol. 1997; 17: 4852-4858Crossref PubMed Scopus (114) Google Scholar). Interaction of Ume6 with Ime1 is required for the phosphorylation of Ume6 at the amino terminus by the Rim11 and Mck1 kinases (33Xiao Y. Mitchell A.P. Mol. Cell. Biol. 2000; 20: 5447-5453Crossref PubMed Scopus (45) Google Scholar). Because Ime1 functions as a transcriptional activator when fused to a DNA-binding domain, it has been proposed that Ime1 provides the activation domain for Ume6 (22Mandel S. Robzyk K. Kassir Y. Dev. Genet. 1994; 15: 139-147Crossref PubMed Scopus (40) Google Scholar).Like with Ume6, phosphorylation of Rgt1 in response to high concentrations of glucose is important for Rgt1 to function as a transcriptional activator. However, it is not known whether phosphorylation facilitates Rgt1 to interact with an activator like Ime1. It is likely that repression by Rgt1 also involves a co-repressor complex like the Sin3 and Rpd3 that mediates deacetylation of histones. Indeed we have preliminary data indicating that glucose induction ofHXT genes involves changes in histone acetylation levels.2 Experiments are under way to test whether the observed changes in histone acetylation at the HXT gene promoters are mediated by Rgt1. In addition, being an activator itself, Ume6 also induces the expression of another transcription factor, Ndt80, that activates middle genes (25Hepworth S.R. Friesen H. Segall J. Mol. Cell. Biol. 1998; 18: 5750-5761Crossref PubMed Google Scholar). Similar to Ume6, Rgt1 is also likely to stimulate the expression of a transcriptional activator that is required for maximal expression of the HXT1 gene in response to high concentrations of glucose (Fig. 7). It has been previously shown that Rgt1 is the target of the glucose induction pathway that leads to up-regulation of HXTgene expression (5Özcan S. Johnston M. Mol. Cell. Biol. 1995; 15: 1564-1572Crossref PubMed Google Scholar). Furthermore, it was demonstrated that Rgt1 is a bifunctional transcription factor that is converted from a transcriptional repressor to an activator in response to high concentrations of glucose (12Özcan S. Leong T. Johnston M. Mol. Cell. Biol. 1996; 16: 6419-6426Crossref PubMed Scopus (134) Google Scholar). In this publication, we have addressed the question of whether glucose regulates the transcriptional activity of Rgt1 by mediating changes in Rgt1 subcellular localization, DNA binding, or post-translational modification. Several transcription factors have been shown to change their subcellular localization in response to external stimuli in yeast, including Mig1 and Pho4 (19DeVit M.J.D. Waddle J.A. Johnston M. Mol. Biol. Cell. 1997; 8: 1603-1618Crossref PubMed Scopus (282) Google Scholar,23Komeili A. O'Shea E.K. Science. 1999; 284: 977-980Crossref PubMed Scopus (245) Google Scholar). Mig1 is phosphorylated by the Snf1 kinase in the absence of glucose or presence of low levels of glucose and is trapped in the cytoplasm. High concentrations of glucose cause dephosphorylation of Mig1 and translocation into the nucleus (19DeVit M.J.D. Waddle J.A. Johnston M. Mol. Biol. Cell. 1997; 8: 1603-1618Crossref PubMed Scopus (282) Google Scholar, 20Treitel M.A. Kuchin S. Carlson M. Mol. Cell. Biol. 1998; 18: 6273-6280Crossref PubMed Scopus (260) Google Scholar). The subcellular localization of Pho4 is regulated in response to phosphate. At high concentrations of phosphate, Pho4 is phosphorylated and exported into the cytoplasm, whereas starvation for phosphate causes dephosphorylation of Pho4 and translocation into the nucleus (23Komeili A. O'Shea E.K. Science. 1999; 284: 977-980Crossref PubMed Scopus (245) Google Scholar). Our data obtained using indirect immunofluorescence microscopy suggest that the subcellular localization of Rgt1 does not change in response to glucose. We also tested the possibility that glucose may regulate the DNA binding affinity of Rgt1 utilizing the ChIP assay. As expected we did not see any significant binding of Rgt1 in vivo to theHXT2 and HXT3 promoters in cells grown in presence of high concentrations of glucose. Rgt1 binding to theHXT2 and HXT3 gene promoters was observed only in the absence of glucose, consistent with its role as a transcriptional repressor of the HXT2 and HXT3 genes when glucose is absent. Several lines of evidence indicate that Rgt1 regulates both repression and activation of HXT1 gene expression. First, Rgt1 is able to bind to a 280-bp promoter region of the HXT1 genein vitro that is sufficient for glucose induction of this gene (12Özcan S. Leong T. Johnston M. Mol. Cell. Biol. 1996; 16: 6419-6426Crossref PubMed Scopus (134) Google Scholar).2 Second, deletion of RGT1 causes a 10-fold increase in HXT1 expression when glucose is absent but a 5–6-fold decrease in HXT1 gene expression at high concentrations of glucose. Third, a lexA-Rgt1 fusion protein functions as a repressor of transcription in the absence of glucose and as an activator of transcription when glucose is abundant (12Özcan S. Leong T. Johnston M. Mol. Cell. Biol. 1996; 16: 6419-6426Crossref PubMed Scopus (134) Google Scholar). These data suggested that Rgt1 might directly regulate HXT1 gene expression in the absence and presence of high concentrations of glucose. However, ChIP analysis of Rgt1 binding in vivoindicates that the positive regulatory effect of Rgt1 onHXT1 gene expression is mediated by an indirect mechanism, because Rgt1 is unable to bind to the HXT1 promoter in response to glucose. It is likely that Rgt1 is required for expression of a transcriptional activator of the HXT1 gene at high concentrations of glucose (Fig. 7). We have consistently observed a change in Rgt1 molecular weight in response to glucose, suggesting that glucose regulates the transcriptional activity of Rgt1 by a post-translational modification event. Treatment of extracts of glucose-grown cells with λ phosphatase indicated that Rgt1 is phosphorylated in response to glucose. Because the molecular mass of the lexA-Rgt1 fusion construct is about 140 kDa, the observed shift in Rgt1 mobility at high concentrations of glucose is likely due to hyperphosphorylation or phosphorylation at multiple sites. The activity of several eukaryotic transcription factors is regulated by reversible phosphorylation in response to changes in the cellular environment. Multisite phosphorylation in particular has been shown to provide a dynamic and precise tuning of the transactivating potential of transcription factors rather than being a static on/off switch. Multisite phosphorylation of transcription factors such as heat shock factor 1 and p53 enables the integration of several different signals (24Holmberg C.I. Tran S.E.F. Eriksson J.E. Sistonen L. Trends Biochem. Sci. 2002; 27: 619-627Abstract Full Text Full Text PDF PubMed Scopus (239) Google Scholar). Multisite phosphorylation of Rgt1 could have several functions such as the disruption of Rgt1 interaction with a repressor complex in the absence of glucose (Fig. 7) and conversion of Rgt1 from a transcriptional repressor to an activator via unmasking of a specific activation domain. Based on several lines of evidence, we propose that Rgt1 functions as a transcriptional repressor of the HXT genes in the absence of glucose, in a complex with the Ssn6-Tup1 and Mth1-Std1 repressor proteins (Fig. 7) (12Özcan S. Leong T. Johnston M. Mol. Cell. Biol. 1996; 16: 6419-6426Crossref PubMed Scopus (134) Google Scholar). 3J. Lakshmanan, A. L. Mosley, and S.Özcan, submitted for publication. When glucose is abundant, Rgt1 becomes hyperphosphorylated and dissociates from the repressor complex. Hyperphosphorylation also converts Rgt1 from a transcriptional repressor to an activator (Fig. 7). Consistent with this idea, deletion of the region from amino acids 75 to 395 abolishes the ability of Rgt1 to both activate transcription and become hyperphosphorylated. Furthermore, the grr1 and snf3 rgt2 mutant strains, where Rgt1 functions always as a transcriptional repressor independent of glucose, lack the glucose-induced modification of Rgt1. The presented data also suggest that Rgt1 is not directly involved in the activation of theHXT1 gene expression but rather stimulates the expression of a transcription factor that is required for maximal expression of theHXT1 gene when glucose is abundant (Fig. 7). The region of Rgt1 required for its glucose-mediated phosphorylation (amino acids 75–395) has several potential phosphorylation sites. Our preliminary data indicate that nuclear localization of Rgt1 is required for the glucose-induced modification, suggesting the idea that the kinase(s) that phosphorylate Rgt1 in response to glucose is nuclear. We are currently testing whether any of the known nuclear kinases are involved in phosphorylation of Rgt1. The transcription factor Ume6 belongs to the same family of DNA-binding proteins as Rgt1 and is also a repressor and an activator of gene expression (26Bowdish K.S. Yuan H.E. Mitchell A.P. Mol. Cell. Biol. 1995; 15: 2955-2961Crossref PubMed Scopus (89) Google Scholar, 27Rubin-Bejerano I. Mandel S. Robzyk K. Kassir Y. Mol. Cell. Biol. 1996; 16: 2518-2526Crossref PubMed Scopus (119) Google Scholar, 28Steber C.M. Esposito R.E. Proc. Natl. Acad. Sci. U. S. A. 1995; 92: 12490-12494Crossref PubMed Scopus (66) Google Scholar). Ume6 in a complex with Sin3 and Rpd3 functions as a repressor; however, its interaction with Ime1 during meiosis converts Ume6 to an activator of early genes (29Washburn B.K. Esposito R.E. Mol. Cell. Biol. 2001; 21: 2057-2069291Crossref PubMed Scopus (75) Google Scholar). The co-repressor Sin3 interacts with the deacetylase Rpd3 and causes repression by deacetylation of histones (30Vidal M. Gaber R.F. Mol. Cell. Biol. 1991; 11: 6317-6327Crossref PubMed Scopus (261) Google Scholar, 31Vidal M. Strich R. Esposito R.E. Gaber R.F. Mol. Cell. Biol. 1991; 11: 6306-6316Crossref PubMed Scopus (142) Google Scholar, 32Kasten M.M. Dorland S. Stillman D.J. Mol. Cell. Biol. 1997; 17: 4852-4858Crossref PubMed Scopus (114) Google Scholar). Interaction of Ume6 with Ime1 is required for the phosphorylation of Ume6 at the amino terminus by the Rim11 and Mck1 kinases (33Xiao Y. Mitchell A.P. Mol. Cell. Biol. 2000; 20: 5447-5453Crossref PubMed Scopus (45) Google Scholar). Because Ime1 functions as a transcriptional activator when fused to a DNA-binding domain, it has been proposed that Ime1 provides the activation domain for Ume6 (22Mandel S. Robzyk K. Kassir Y. Dev. Genet. 1994; 15: 139-147Crossref PubMed Scopus (40) Google Scholar). Like with Ume6, phosphorylation of Rgt1 in response to high concentrations of glucose is important for Rgt1 to function as a transcriptional activator. However, it is not known whether phosphorylation facilitates Rgt1 to interact with an activator like Ime1. It is likely that repression by Rgt1 also involves a co-repressor complex like the Sin3 and Rpd3 that mediates deacetylation of histones. Indeed we have preliminary data indicating that glucose induction ofHXT genes involves changes in histone acetylation levels.2 Experiments are under way to test whether the observed changes in histone acetylation at the HXT gene promoters are mediated by Rgt1. In addition, being an activator itself, Ume6 also induces the expression of another transcription factor, Ndt80, that activates middle genes (25Hepworth S.R. Friesen H. Segall J. Mol. Cell. Biol. 1998; 18: 5750-5761Crossref PubMed Google Scholar). Similar to Ume6, Rgt1 is also likely to stimulate the expression of a transcriptional activator that is required for maximal expression of the HXT1 gene in response to high concentrations of glucose (Fig. 7). We thank Mark Johnston and the members of his laboratory for sharing data prior to publication and Bob Dickson and Bob Lester for useful suggestions. We thank Dr. Wally Whiteheart for providing us with HA antibodies and Courtney Reynolds for excellent technical assistance.
Referência(s)