Sfl1 functions via the co-repressor Ssn6-Tup1 and the cAMP-dependent protein kinase Tpk2

J Mol Biol. 2001 Jun 22;309(5):1007-15. doi: 10.1006/jmbi.2001.4742.

Abstract

Ssn6 (Cyc8) is a component of the yeast general corepressor Ssn6-Tup1 that inhibits the transcription of many diversely regulated genes. The corepressor does not interact directly with DNA but is recruited to different promoters through interactions with distinct pathway-specific, DNA-binding repressor proteins. Using yeast two-hybrid and GST chromatography interaction experiments, we have determined that Sfl1, a novel repressor protein, interacts directly with Ssn6, and in vivo repression data suggest that Sfl1 inhibits transcription by recruiting Ssn6-Tup1 via a specific domain in the Sfl1 protein. Sin4 and Srb10, components of specific RNA polymerase II sub-complexes that are required for Ssn6-Tup1 repression activity, are found to be required for Sfl1 repression function. These results indicate a possible mechanism for Sfl1-mediated repression via Ssn6-Tup1 and specific subunits of the RNA polymerase II holoenzyme. Electrophoretic mobility shift and chromatin immuno-precipitation assays demonstrate that Sfl1 is present at the promoters of three Ssn6-Tup1-repressible genes; namely, FLO11, HSP26, and SUC2. Sfl1 is known to interact with Tpk2, a cAMP-dependent protein kinase that negatively regulates Sfl1 function. Consistently, we show that phosphorylation by protein kinase A inhibits Sfl1 DNA binding in vitro, and that a tpk2Delta mutation increases the levels of Sfl1 protein associated with specific promoter elements in vivo. These data indicate a possible mechanism for regulating Sfl1-mediated repression through modulation of DNA binding by cAMP-dependent protein kinase-dependent phosphorylation. Taken together with previous data, these new observations suggest a link between cAMP signaling and Ssn6-Tup1-mediated transcriptional repression.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Bacterial Proteins / genetics
  • Bacterial Proteins / metabolism
  • Chromatin / genetics
  • Chromatin / metabolism
  • Cyclic AMP / pharmacology*
  • Cyclic AMP-Dependent Protein Kinases / chemistry
  • Cyclic AMP-Dependent Protein Kinases / metabolism
  • Cyclin-Dependent Kinase 8
  • Cyclin-Dependent Kinases / genetics
  • Cyclin-Dependent Kinases / metabolism
  • DNA, Fungal / genetics
  • DNA, Fungal / metabolism
  • DNA-Binding Proteins*
  • Fungal Proteins / chemistry
  • Fungal Proteins / genetics
  • Fungal Proteins / metabolism*
  • Gene Expression Regulation, Fungal
  • Holoenzymes / chemistry
  • Holoenzymes / metabolism
  • Mediator Complex
  • Membrane Glycoproteins
  • Membrane Proteins / genetics
  • Mutation / genetics
  • Nuclear Proteins*
  • Phosphorylation / drug effects
  • Precipitin Tests
  • Promoter Regions, Genetic / genetics
  • Protein Binding
  • Protein Kinases / metabolism*
  • RNA Polymerase II / chemistry
  • RNA Polymerase II / genetics
  • RNA Polymerase II / metabolism
  • Repressor Proteins / genetics
  • Repressor Proteins / metabolism*
  • Saccharomyces cerevisiae / drug effects
  • Saccharomyces cerevisiae / enzymology
  • Saccharomyces cerevisiae / genetics
  • Saccharomyces cerevisiae / metabolism*
  • Saccharomyces cerevisiae Proteins*
  • Serine Endopeptidases / genetics
  • Serine Endopeptidases / metabolism
  • Signal Transduction / drug effects
  • Trans-Activators*
  • Transcription Factors*
  • Transcription, Genetic / genetics
  • Two-Hybrid System Techniques

Substances

  • Bacterial Proteins
  • CYC8 protein, S cerevisiae
  • Chromatin
  • DNA, Fungal
  • DNA-Binding Proteins
  • FLO11 protein, S cerevisiae
  • Fungal Proteins
  • Holoenzymes
  • LexA protein, Bacteria
  • Mediator Complex
  • Membrane Glycoproteins
  • Membrane Proteins
  • Nuclear Proteins
  • Repressor Proteins
  • SFL1 protein, S cerevisiae
  • SIN4 protein, S cerevisiae
  • Saccharomyces cerevisiae Proteins
  • TUP1 protein, S cerevisiae
  • Trans-Activators
  • Transcription Factors
  • Cyclic AMP
  • Protein Kinases
  • Cyclic AMP-Dependent Protein Kinases
  • Cyclin-Dependent Kinase 8
  • Cyclin-Dependent Kinases
  • SSN3 protein, S cerevisiae
  • RNA Polymerase II
  • Serine Endopeptidases