The Snf1 protein kinase and Sit4 protein phosphatase have opposing functions in regulating TATA-binding protein association with the Saccharomyces cerevisiae INO1 promoter

Genetics. 2005 Apr;169(4):1957-72. doi: 10.1534/genetics.104.038075. Epub 2005 Feb 16.

Abstract

To identify the mechanisms by which multiple signaling pathways coordinately affect gene expression, we investigated regulation of the S. cerevisiae INO1 gene. Full activation of INO1 transcription occurs in the absence of inositol and requires the Snf1 protein kinase in addition to other signaling molecules and transcription factors. Here, we present evidence that the Sit4 protein phosphatase negatively regulates INO1 transcription. A mutation in SIT4 was uncovered as a suppressor of the inositol auxotrophy of snf1Delta strains. We found that sit4 mutant strains exhibit an Spt(-) phenotype, suggesting a more general role for Sit4 in transcription. In fact, like the gene-specific regulators of INO1 transcription, Opi1, Ino2, and Ino4, both Snf1 and Sit4 regulate binding of TBP to the INO1 promoter, as determined by chromatin immunoprecipitation analysis. Experiments involving double-mutant strains indicate that the negative effect of Sit4 on INO1 transcription is unlikely to occur through dephosphorylation of histone H3 or Opi1. Sit4 is a known component of the target of rapamycin (TOR) signaling pathway, and treatment of cells with rapamycin reduces INO1 activation. However, analysis of rapamycin-treated cells suggests that Sit4 represses INO1 transcription through multiple mechanisms, only one of which may involve inhibition of TOR signaling.

Publication types

  • Research Support, N.I.H., Extramural
  • Research Support, U.S. Gov't, P.H.S.

MeSH terms

  • Alleles
  • Chromatin Immunoprecipitation
  • Cloning, Molecular
  • Codon, Nonsense
  • DNA Primers / chemistry
  • Gene Expression Regulation, Fungal*
  • Genotype
  • Histones / metabolism
  • Immunoblotting
  • Lac Operon
  • Models, Genetic
  • Mutation
  • Myo-Inositol-1-Phosphate Synthase / genetics*
  • Phenotype
  • Phosphoprotein Phosphatases / genetics*
  • Phosphoprotein Phosphatases / physiology*
  • Phosphorylation
  • Plasmids / metabolism
  • Protein Binding
  • Protein Kinases / pharmacology
  • Protein Phosphatase 2
  • Protein-Serine-Threonine Kinases / genetics*
  • Protein-Serine-Threonine Kinases / physiology*
  • Repressor Proteins / metabolism
  • Saccharomyces cerevisiae / genetics*
  • Saccharomyces cerevisiae / physiology*
  • Saccharomyces cerevisiae Proteins / metabolism
  • Signal Transduction
  • Sirolimus / pharmacology
  • TATA-Box Binding Protein / metabolism*
  • TOR Serine-Threonine Kinases
  • Transcription, Genetic
  • beta-Galactosidase / metabolism

Substances

  • Codon, Nonsense
  • DNA Primers
  • Histones
  • OPI1 protein, S cerevisiae
  • Repressor Proteins
  • Saccharomyces cerevisiae Proteins
  • TATA-Box Binding Protein
  • Protein Kinases
  • SNF1-related protein kinases
  • TOR Serine-Threonine Kinases
  • Protein-Serine-Threonine Kinases
  • Phosphoprotein Phosphatases
  • Protein Phosphatase 2
  • SIT4 protein, S cerevisiae
  • beta-Galactosidase
  • Myo-Inositol-1-Phosphate Synthase
  • Sirolimus