Sfl1 functions via the co-repressor Ssn6-Tup1 and the cAMP-dependent protein kinase Tpk2.
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.
Conlan
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RS
Institute of Molecular Biology & Biotechnology-Foundation of Research & Technology, Vassilika Vouton, Heraklion, Crete, GR-711 10, Greece. r.s.conlan@swan.ac.uk
Tzamarias
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D
D001426:Bacterial Proteins / Q000235:genetics / Q000378:metabolism; D002843:Chromatin / Q000235:genetics / Q000378:metabolism; D000242:Cyclic AMP / Q000494:pharmacology*; D017868:Cyclic AMP-Dependent Protein Kinases / Q000737:chemistry / Q000378:metabolism; D056850:Cyclin-Dependent Kinase 8; D018844:Cyclin-Dependent Kinases / Q000235:genetics / Q000378:metabolism; D004271:DNA, Fungal / Q000235:genetics / Q000378:metabolism; D004268:DNA-Binding Proteins; D005656:Fungal Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D015966:Gene Expression Regulation, Fungal; D020035:Holoenzymes / Q000737:chemistry / Q000378:metabolism; D056892:Mediator Complex; D008562:Membrane Glycoproteins; D008565:Membrane Proteins / Q000235:genetics; D009154:Mutation / Q000235:genetics; D009687:Nuclear Proteins; D010766:Phosphorylation / Q000187:drug effects; D011233:Precipitin Tests; D011401:Promoter Regions, Genetic / Q000235:genetics; D011485:Protein Binding; D011494:Protein Kinases / Q000378:metabolism*; D012319:RNA Polymerase II / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D012097:Repressor Proteins / Q000235:genetics / Q000378:metabolism*; D012441:Saccharomyces cerevisiae / Q000187:drug effects / Q000201:enzymology / Q000235:genetics / Q000378:metabolism*; D029701:Saccharomyces cerevisiae Proteins; D012697:Serine Endopeptidases / Q000235:genetics / Q000378:metabolism; D015398:Signal Transduction / Q000187:drug effects; D015534:Trans-Activators; D014157:Transcription Factors; D014158:Transcription, Genetic / Q000235:genetics; D020798:Two-Hybrid System Techniques; D000099160:LexA Repressor Protein
D001426:Bacterial Proteins; D002843:Chromatin; D000242:Cyclic AMP; D017868:Cyclic AMP-Dependent Protein Kinases; D056850:Cyclin-Dependent Kinase 8; D018844:Cyclin-Dependent Kinases; D004271:DNA, Fungal; D004268:DNA-Binding Proteins; D005656:Fungal Proteins; D020035:Holoenzymes; D056892:Mediator Complex; D008562:Membrane Glycoproteins; D008565:Membrane Proteins; D009687:Nuclear Proteins; D011494:Protein Kinases; D012319:RNA Polymerase II; D012097:Repressor Proteins; D029701:Saccharomyces cerevisiae Proteins; D012697:Serine Endopeptidases; D015534:Trans-Activators; D014157:Transcription Factors; D000099160:LexA Repressor Protein; C065455:CYC8 protein, S cerevisiae; C110690:FLO11 protein, S cerevisiae; C063404:SFL1 protein, S cerevisiae; C076878:SIN4 protein, S cerevisiae; C066502:TUP1 protein, S cerevisiae; C498309:SSN3 protein, S cerevisiae