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.
Copyright 2001 Academic Press.
Publication types
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Research Support, Non-U.S. Gov't
MeSH terms
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Bacterial Proteins / genetics
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Bacterial Proteins / metabolism
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Chromatin / genetics
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Chromatin / metabolism
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Cyclic AMP / pharmacology*
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Cyclic AMP-Dependent Protein Kinases / chemistry
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Cyclic AMP-Dependent Protein Kinases / metabolism
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Cyclin-Dependent Kinase 8
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Cyclin-Dependent Kinases / genetics
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Cyclin-Dependent Kinases / metabolism
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DNA, Fungal / genetics
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DNA, Fungal / metabolism
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DNA-Binding Proteins*
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Fungal Proteins / chemistry
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Fungal Proteins / genetics
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Fungal Proteins / metabolism*
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Gene Expression Regulation, Fungal
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Holoenzymes / chemistry
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Holoenzymes / metabolism
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Mediator Complex
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Membrane Glycoproteins
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Membrane Proteins / genetics
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Mutation / genetics
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Nuclear Proteins*
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Phosphorylation / drug effects
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Precipitin Tests
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Promoter Regions, Genetic / genetics
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Protein Binding
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Protein Kinases / metabolism*
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RNA Polymerase II / chemistry
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RNA Polymerase II / genetics
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RNA Polymerase II / metabolism
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Repressor Proteins / genetics
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Repressor Proteins / metabolism*
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Saccharomyces cerevisiae / drug effects
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Saccharomyces cerevisiae / enzymology
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Saccharomyces cerevisiae / genetics
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Saccharomyces cerevisiae / metabolism*
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Saccharomyces cerevisiae Proteins*
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Serine Endopeptidases / genetics
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Serine Endopeptidases / metabolism
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Signal Transduction / drug effects
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Trans-Activators*
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Transcription Factors*
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Transcription, Genetic / genetics
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Two-Hybrid System Techniques
Substances
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Bacterial Proteins
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CYC8 protein, S cerevisiae
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Chromatin
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DNA, Fungal
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DNA-Binding Proteins
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FLO11 protein, S cerevisiae
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Fungal Proteins
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Holoenzymes
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LexA protein, Bacteria
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Mediator Complex
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Membrane Glycoproteins
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Membrane Proteins
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Nuclear Proteins
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Repressor Proteins
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SFL1 protein, S cerevisiae
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SIN4 protein, S cerevisiae
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Saccharomyces cerevisiae Proteins
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TUP1 protein, S cerevisiae
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Trans-Activators
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Transcription Factors
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Cyclic AMP
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Protein Kinases
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Cyclic AMP-Dependent Protein Kinases
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Cyclin-Dependent Kinase 8
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Cyclin-Dependent Kinases
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SSN3 protein, S cerevisiae
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RNA Polymerase II
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Serine Endopeptidases