Histone H3 Ser10 Phosphorylation-Independent Function of Snf1 and Reg1 Proteins Rescues a gcn5− Mutant in HIS3 Expression

ABSTRACT Gcn5 protein is a prototypical histone acetyltransferase that controls transcription of multiple yeast genes. To identify molecular functions that act downstream of or in parallel with Gcn5 protein, we screened for suppressors that rescue the transcriptional defects of HIS3 caused by a catalytically inactive mutant Gcn5, the E173H mutant. One bypass of Gcn5 requirement gene (BGR) suppressor was mapped to the REG1 locus that encodes a semidominant mutant truncated after amino acid 740. Reg1(1-740) protein does not rescue the complete knockout of GCN5, nor does it suppress other gcn5 − defects, including the inability to utilize nonglucose carbon sources. Reg1(1-740) enhances HIS3 transcription while HIS3 promoter remains hypoacetylated, indicating that a noncatalytic function of Gcn5 is targeted by this suppressor protein. Reg1 protein is a major regulator of Snf1 kinase that phosphorylates Ser10 of histone H3. However, whereas Snf1 protein is important for HIS3 expression, replacing Ser10 of H3 with alanine or glutamate neither attenuates nor augments the BGR phenotypes. Overproduction of Snf1 protein also preferentially rescues the E173H allele. Biochemically, both Snf1 and Reg1(1-740) proteins copurify with Gcn5 protein. Snf1 can phosphorylate recombinant Gcn5 in vitro. Together, these data suggest that Reg1 and Snf1 proteins function in an H3 phosphorylation-independent pathway that also involves a noncatalytic role played by Gcn5 protein.

[1]  M. Carlson,et al.  The AMP-activated/SNF1 protein kinase subfamily: metabolic sensors of the eukaryotic cell? , 1998, Annual review of biochemistry.

[2]  John R Yates,et al.  A Subset of TAFIIs Are Integral Components of the SAGA Complex Required for Nucleosome Acetylation and Transcriptional Stimulation , 1998, Cell.

[3]  J. Jaehning,et al.  Glucose repression of yeast mitochondrial transcription: kinetics of derepression and role of nuclear genes , 1994, Molecular and cellular biology.

[4]  Ronen Marmorstein,et al.  Structural basis for histone and phosphohistone binding by the GCN5 histone acetyltransferase. , 2003, Molecular cell.

[5]  John R. Yates,et al.  The Novel SLIK Histone Acetyltransferase Complex Functions in the Yeast Retrograde Response Pathway , 2002, Molecular and Cellular Biology.

[6]  K. Struhl,et al.  Gcn4 activator targets Gcn5 histone acetyltransferase to specific promoters independently of transcription. , 2000, Molecular cell.

[7]  B. Pugh,et al.  A genome-wide housekeeping role for TFIID and a highly regulated stress-related role for SAGA in Saccharomyces cerevisiae. , 2004, Molecular cell.

[8]  F. Winston,et al.  Essential functional interactions of SAGA, a Saccharomyces cerevisiae complex of Spt, Ada, and Gcn5 proteins, with the Snf/Swi and Srb/mediator complexes. , 1997, Genetics.

[9]  F. Winston,et al.  The S. cerevisiae SAGA complex functions in vivo as a coactivator for transcriptional activation by Gal4. , 2001, Genes & development.

[10]  Hans-Joachim Schüller,et al.  Transcriptional control of nonfermentative metabolism in the yeast Saccharomyces cerevisiae , 2003, Current Genetics.

[11]  S. Berger,et al.  Critical residues for histone acetylation by Gcn5, functioning in Ada and SAGA complexes, are also required for transcriptional function in vivo. , 1998, Genes & development.

[12]  M. Goebl,et al.  Genetic interactions between REG1/HEX2 and GLC7, the gene encoding the protein phosphatase type 1 catalytic subunit in Saccharomyces cerevisiae. , 1996, Genetics.

[13]  K. Entian,et al.  Characterization of Hex2 protein, a negative regulatory element necessary for glucose repression in yeast. , 1991, European journal of biochemistry.

[14]  K. M. Dombek,et al.  The Reg1-interacting Proteins, Bmh1, Bmh2, Ssb1, and Ssb2, Have Roles in Maintaining Glucose Repression in Saccharomyces cerevisiae* , 2004, Journal of Biological Chemistry.

[15]  J. W. Rooney,et al.  SPT3 interacts with TFIID to allow normal transcription in Saccharomyces cerevisiae. , 1992, Genes & development.

[16]  D. Stillman,et al.  Regulation of TATA-Binding Protein Binding by the SAGA Complex and the Nhp6 High-Mobility Group Protein , 2003, Molecular and Cellular Biology.

[17]  J Wu,et al.  Multiple regulatory proteins mediate repression and activation by interaction with the yeast Mig1 binding site , 1998, Yeast.

[18]  S. Berger,et al.  Snf1--a Histone Kinase That Works in Concert with the Histone Acetyltransferase Gcn5 to Regulate Transcription , 2001, Science.

[19]  C. Allis,et al.  Solution structure of the catalytic domain of GCN5 histone acetyltransferase bound to coenzyme A , 1999, Nature.

[20]  F. Winston,et al.  Evidence That Spt10 and Spt21 of Saccharomyces cerevisiae Play Distinct Roles in Vivo and Functionally Interact With MCB-Binding Factor, SCB-Binding Factor and Snf1 , 2005, Genetics.

[21]  G. Fink,et al.  Methods in yeast genetics , 1979 .

[22]  M. Green,et al.  SAGA is an essential in vivo target of the yeast acidic activator Gal4p. , 2001, Genes & development.

[23]  J. Smith,et al.  lacZY gene fusion cassettes with KanR resistance. , 1988, Nucleic acids research.

[24]  L. Prakash,et al.  Yeast Saccharomyces cerevisiae selectable markers in pUC18 polylinkers , 1990, Yeast.

[25]  C. Allis,et al.  Histone acetyltransferases. , 2001, Annual review of biochemistry.

[26]  K. Arndt,et al.  Evidence for the involvement of the Glc7-Reg1 phosphatase and the Snf1-Snf4 kinase in the regulation of INO1 transcription in Saccharomyces cerevisiae. , 1999, Genetics.

[27]  R. Tjian,et al.  Bromodomains mediate an acetyl-histone encoded antisilencing function at heterochromatin boundaries. , 2003, Molecular cell.

[28]  C. Peterson,et al.  Functional interaction between GCN5 and polyamines: a new role for core histone acetylation , 1999, The EMBO journal.

[29]  D. Sterner,et al.  SALSA, a variant of yeast SAGA, contains truncated Spt7, which correlates with activated transcription , 2002, Proceedings of the National Academy of Sciences of the United States of America.

[30]  M. Carlson,et al.  Glucose-regulated interaction of a regulatory subunit of protein phosphatase 1 with the Snf1 protein kinase in Saccharomyces cerevisiae. , 1998, Proceedings of the National Academy of Sciences of the United States of America.

[31]  C. Allis,et al.  Cell cycle-regulated histone acetylation required for expression of the yeast HO gene. , 1999, Genes & development.

[32]  Lei Zeng,et al.  Structure and ligand of a histone acetyltransferase bromodomain , 1999, Nature.

[33]  L. Pillus,et al.  Molecular Requirements for Gene Expression Mediated by Targeted Histone Acetyltransferases , 2004, Molecular and Cellular Biology.

[34]  P. Philippsen,et al.  New heterologous modules for classical or PCR‐based gene disruptions in Saccharomyces cerevisiae , 1994, Yeast.

[35]  Timothy A. J. Haystead,et al.  Regulatory Interactions between the Reg1-Glc7 Protein Phosphatase and the Snf1 Protein Kinase , 2000, Molecular and Cellular Biology.

[36]  M. Ajimura,et al.  GCN5-dependent histone H3 acetylation and RPD3-dependent histone H4 deacetylation have distinct, opposing effects on IME2 transcription, during meiosis and during vegetative growth, in budding yeast. , 1999, Proceedings of the National Academy of Sciences of the United States of America.

[37]  Yong-Yeon Cho,et al.  Phosphorylation of Ser28 in Histone H3 Mediated by Mixed Lineage Kinase-like Mitogen-activated Protein Triple Kinase α* , 2005, Journal of Biological Chemistry.

[38]  M. Carlson,et al.  Protein phosphatase type 1 interacts with proteins required for meiosis and other cellular processes in Saccharomyces cerevisiae , 1996, Molecular and cellular biology.

[39]  M. Carlson,et al.  REG1 binds to protein phosphatase type 1 and regulates glucose repression in Saccharomyces cerevisiae. , 1995, The EMBO journal.

[40]  R. Schiestl,et al.  Improved method for high efficiency transformation of intact yeast cells. , 1992, Nucleic acids research.

[41]  C. Allis,et al.  Synergistic coupling of histone H3 phosphorylation and acetylation in response to epidermal growth factor stimulation. , 2000, Molecular cell.

[42]  Kenneth M. Dombek,et al.  Functional Analysis of the Yeast Glc7-Binding Protein Reg1 Identifies a Protein Phosphatase Type 1-Binding Motif as Essential for Repression of ADH2 Expression , 1999, Molecular and Cellular Biology.

[43]  M. Carlson,et al.  Molecular analysis of the SNF4 gene of Saccharomyces cerevisiae: evidence for physical association of the SNF4 protein with the SNF1 protein kinase , 1989, Molecular and cellular biology.

[44]  P. Ross-Macdonald,et al.  Transposon mutagenesis for the analysis of protein production, function, and localization. , 1999, Methods in enzymology.

[45]  Jacques Côté,et al.  The diverse functions of histone acetyltransferase complexes. , 2003, Trends in genetics : TIG.

[46]  M. Carlson,et al.  A protein kinase substrate identified by the two-hybrid system. , 1992, Science.

[47]  Sung-Hee Ahn,et al.  Sterile 20 Kinase Phosphorylates Histone H2B at Serine 10 during Hydrogen Peroxide-Induced Apoptosis in S. cerevisiae , 2005, Cell.

[48]  M. Johnston,et al.  Genetic and molecular characterization of GAL83: its interaction and similarities with other genes involved in glucose repression in Saccharomyces cerevisiae. , 1993, Genetics.

[49]  Fred Winston,et al.  Functional Organization of the Yeast SAGA Complex: Distinct Components Involved in Structural Integrity, Nucleosome Acetylation, and TATA-Binding Protein Interaction , 1999, Molecular and Cellular Biology.

[50]  M. Inagaki,et al.  Mitosis-specific histone H3 phosphorylation in vitro in nucleosome structures. , 1990, European journal of biochemistry.

[51]  B. Zhang,et al.  Analysis of the mechanism by which glucose inhibits maltose induction of MAL gene expression in Saccharomyces. , 2000, Genetics.

[52]  A. Hopper,et al.  SRN1, a yeast gene involved in RNA processing, is identical to HEX2/REG1, a negative regulator in glucose repression , 1992, Molecular and cellular biology.

[53]  A. Dudley,et al.  The Spt components of SAGA facilitate TBP binding to a promoter at a post-activator-binding step in vivo. , 1999, Genes & development.

[54]  E. W. Jones,et al.  Regulation of the proteinase B structural gene PRB1 in Saccharomyces cerevisiae , 1997, Journal of bacteriology.

[55]  M. Carlson,et al.  A regulatory shortcut between the Snf1 protein kinase and RNA polymerase II holoenzyme. , 2000, Proceedings of the National Academy of Sciences of the United States of America.

[56]  K. M. Dombek,et al.  ADH2 expression is repressed by REG1 independently of mutations that alter the phosphorylation of the yeast transcription factor ADR1 , 1993, Molecular and cellular biology.

[57]  R Ohba,et al.  Yeast Gcn5 functions in two multisubunit complexes to acetylate nucleosomal histones: characterization of an Ada complex and the SAGA (Spt/Ada) complex. , 1997, Genes & development.

[58]  A. Schmid,et al.  Increasing the rate of chromatin remodeling and gene activation—a novel role for the histone acetyltransferase Gcn5 , 2001, The EMBO journal.

[59]  J. Denu,et al.  Mutational Analysis of Conserved Residues in the GCN5 Family of Histone Acetyltransferases* , 2001, The Journal of Biological Chemistry.

[60]  T. Hughes,et al.  H2B Ubiquitin Protease Ubp8 and Sgf11 Constitute a Discrete Functional Module within the Saccharomyces cerevisiae SAGA Complex , 2005, Molecular and Cellular Biology.

[61]  M. Brand,et al.  Three-dimensional structures of the TAFII-containing complexes TFIID and TFTC. , 1999, Science.

[62]  Ronen Marmorstein,et al.  Structure of Tetrahymena GCN5 bound to coenzyme A and a histone H3 peptide , 1999, Nature.

[63]  Patrick Schultz,et al.  Molecular architecture of the S. cerevisiae SAGA complex. , 2004, Molecular cell.

[64]  S. Berger,et al.  Phosphorylation of serine 10 in histone H3 is functionally linked in vitro and in vivo to Gcn5-mediated acetylation at lysine 14. , 2000, Molecular cell.

[65]  S. Buratowski,et al.  Different sensitivities of bromodomain factors 1 and 2 to histone H4 acetylation. , 2003, Molecular cell.

[66]  K. Tatchell,et al.  Protein phosphatase type 1 regulates ion homeostasis in Saccharomyces cerevisiae. , 2002, Genetics.

[67]  J. Boeke,et al.  Designer deletion strains derived from Saccharomyces cerevisiae S288C: A useful set of strains and plasmids for PCR‐mediated gene disruption and other applications , 1998, Yeast.

[68]  K. Tatchell,et al.  The REG2 gene of Saccharomyces cerevisiae encodes a type 1 protein phosphatase-binding protein that functions with Reg1p and the Snf1 protein kinase to regulate growth , 1996, Molecular and cellular biology.

[69]  M. Carlson,et al.  Srb/mediator proteins interact functionally and physically with transcriptional repressor Sfl1 , 1998, The EMBO journal.

[70]  S. Fields,et al.  A tethered catalysis, two-hybrid system to identify protein-protein interactions requiring post-translational modifications , 2004, Nature Biotechnology.

[71]  D. Sterner,et al.  Inhibition of TATA-Binding Protein Function by SAGA Subunits Spt3 and Spt8 at Gcn4-Activated Promoters , 2000, Molecular and Cellular Biology.

[72]  H. Reinke,et al.  Multiple Mechanistically Distinct Functions of SAGA at the PHO5 Promoter , 2003, Molecular and Cellular Biology.

[73]  K. Natarajan,et al.  An Array of Coactivators Is Required for Optimal Recruitment of TATA Binding Protein and RNA Polymerase II by Promoter-Bound Gcn4p , 2004, Molecular and Cellular Biology.

[74]  M. Paul,et al.  Highly conserved protein kinases involved in the regulation of carbon and amino acid metabolism. , 2003, Journal of experimental botany.

[75]  Michael R. Green,et al.  Differential Requirement of SAGA Components for Recruitment of TATA-Box-Binding Protein to Promoters In Vivo , 2002, Molecular and Cellular Biology.

[76]  R. Tjian,et al.  Structure and function of a human TAFII250 double bromodomain module. , 2000, Science.

[77]  Erich A Nigg,et al.  Aurora‐B phosphorylates Histone H3 at serine28 with regard to the mitotic chromosome condensation , 2002, Genes to cells : devoted to molecular & cellular mechanisms.

[78]  S. Berger,et al.  Crystal structure of the histone acetyltransferase domain of the human PCAF transcriptional regulator bound to coenzyme A , 1999, The EMBO journal.

[79]  A. Carr,et al.  The 14-3-3 proteins encoded by the BMH1 and BMH2 genes are essential in the yeast Saccharomyces cerevisiae and can be replaced by a plant homologue. , 1995, European journal of biochemistry.

[80]  M. Johnston,et al.  Two systems of glucose repression of the GAL1 promoter in Saccharomyces cerevisiae , 1990, Molecular and cellular biology.

[81]  C. Peterson,et al.  Histones and histone modifications , 2004, Current Biology.

[82]  M. Carlson,et al.  Mutations causing constitutive invertase synthesis in yeast: genetic interactions with snf mutations. , 1987, Genetics.

[83]  D. Sterner,et al.  Crystal structure and mechanism of histone acetylation of the yeast GCN5 transcriptional coactivator. , 1999, Proceedings of the National Academy of Sciences of the United States of America.

[84]  A. Neiman,et al.  A Gip1p–Glc7p phosphatase complex regulates septin organization and spore wall formation , 2001, The Journal of cell biology.

[85]  C. Peterson,et al.  Role for ADA/GCN5 products in antagonizing chromatin-mediated transcriptional repression , 1997, Molecular and cellular biology.

[86]  John R Yates,et al.  Deubiquitination of Histone H2B by a Yeast Acetyltransferase Complex Regulates Transcription* , 2004, Journal of Biological Chemistry.

[87]  B. Séraphin,et al.  A generic protein purification method for protein complex characterization and proteome exploration , 1999, Nature Biotechnology.

[88]  S. Berger,et al.  Catalytic Mechanism and Function of Invariant Glutamic Acid 173 from the Histone Acetyltransferase GCN5 Transcriptional Coactivator* , 1999, The Journal of Biological Chemistry.

[89]  C. Brandl,et al.  Components of the SAGA Histone Acetyltransferase Complex Are Required for Repressed Transcription of ARG1 in Rich Medium , 2002, Molecular and Cellular Biology.

[90]  Ali Shilatifard,et al.  Transcriptional activation via sequential histone H2B ubiquitylation and deubiquitylation, mediated by SAGA-associated Ubp8. , 2003, Genes & development.

[91]  J. Denu,et al.  Kinetic Mechanism of the Histone Acetyltransferase GCN5 from Yeast* , 2000, The Journal of Biological Chemistry.

[92]  F. Sherman.  Getting started with yeast. , 1991, Methods in enzymology.

[93]  S. Berger,et al.  Histone H3 phosphorylation can promote TBP recruitment through distinct promoter‐specific mechanisms , 2005, The EMBO journal.

[94]  M. Carlson,et al.  Snf1 Protein Kinase Regulates Phosphorylation of the Mig1 Repressor in Saccharomyces cerevisiae , 1998, Molecular and Cellular Biology.

[95]  Michael R. Green,et al.  Dissecting the Regulatory Circuitry of a Eukaryotic Genome , 1998, Cell.

[96]  John R. Yates,et al.  Chd1 chromodomain links histone H3 methylation with SAGA- and SLIK-dependent acetylation , 2005, Nature.

[97]  C. Allis,et al.  Roles of histone acetyltransferases and deacetylases in gene regulation , 1998, BioEssays : news and reviews in molecular, cellular and developmental biology.

[98]  R. D. Gietz,et al.  New yeast-Escherichia coli shuttle vectors constructed with in vitro mutagenized yeast genes lacking six-base pair restriction sites. , 1988, Gene.

[99]  K. Matsumoto,et al.  Recessive mutations conferring resistance to carbon catabolite repression of galactokinase synthesis in Saccharomyces cerevisiae , 1983, Journal of bacteriology.

[100]  J. Gordon,et al.  Sip2, an N-Myristoylated β Subunit of Snf1 Kinase, Regulates Aging in Saccharomyces cerevisiae by Affecting Cellular Histone Kinase Activity, Recombination at rDNA Loci, and Silencing* , 2003, The Journal of Biological Chemistry.

[101]  C. Allis,et al.  Histone acetyltransferase activity of yeast Gcn5p is required for the activation of target genes in vivo. , 1998, Genes & development.

[102]  Làszlò Tora,et al.  SAGA unveiled. , 2005, Trends in biochemical sciences.

[103]  R. Tjian,et al.  Three-dimensional structure of the human TFIID-IIA-IIB complex. , 1999, Science.

[104]  J. Hahn,et al.  Activation of the Saccharomyces cerevisiae Heat Shock Transcription Factor Under Glucose Starvation Conditions by Snf1 Protein Kinase* , 2004, Journal of Biological Chemistry.

[105]  S. Berger,et al.  Repression of GCN5 Histone Acetyltransferase Activity via Bromodomain-Mediated Binding and Phosphorylation by the Ku–DNA-Dependent Protein Kinase Complex , 1998, Molecular and Cellular Biology.