title: Recruitment of Tup1-Ssn6 by yeast hypoxic genes and chromatin-independent exclusion of TATA binding protein.
abstract: The Tup1-Ssn6 general repression complex in Saccharomyces cerevisiae represses a wide variety of regulons. Regulon-specific DNA binding proteins recruit the repression complex, and their synthesis, activity, or localization controls the conditions for repression. Rox1 is the hypoxic regulon-specific protein, and a second DNA binding protein, Mot3, augments repression at tightly controlled genes. We addressed the requirements for Tup1-Ssn6 recruitment to two hypoxic genes, ANB1 and HEM13, by using chromatin immunoprecipitation assays. Either Rox1 or Mot3 could recruit Ssn6, but Tup1 recruitment required Ssn6 and Rox1. We also monitored events during derepression. Rox1 and Mot3 dissociated from DNA quickly, accounting for the rapid accumulation of ANB1 and HEM13 RNAs, suggesting a simple explanation for induction. However, Tup1 remained associated with these genes, suggesting that the localization of Tup1-Ssn6 is not the sole determinant of repression. We could not reproduce the observation that deletion of the Tup1-Ssn6-interacting protein Cti6 was required for induction. Finally, Tup1 is capable of repression through a chromatin-dependent mechanism, the positioning of a nucleosome over the TATA box, or a chromatin-independent mechanism. We found that the rate of derepression was independent of the positioned nucleosome and that the TATA binding protein was excluded from ANB1 even in the absence of the positioned nucleosome. The mediator factor Srb7 has been shown to interact with Tup1 and to play a role in repression at several regulons, but we found that significant levels of repression remained in srb7 mutants even when the chromatin-dependent repression mechanism was eliminated. These findings suggest that the repression of different regulons or genes may invoke different mechanisms.
authors.affiliation: Department of Biological Sciences, State University of New York at Albany, Albany, New York 12222, USA.
authors.forename: Thomas A
authors.initials: TA
authors.lastname: Mennella
authors.forename: Lee G
authors.initials: LG
authors.lastname: Klinkenberg
authors.forename: Richard S
authors.initials: RS
authors.lastname: Zitomer
mesh_terms: D015687:Cell Hypoxia; D002843:Chromatin / Q000737:chemistry* / Q000378:metabolism; D004268:DNA-Binding Proteins / Q000235:genetics / Q000378:metabolism; D005656:Fungal Proteins / Q000235:genetics / Q000378:metabolism; D005800:Genes, Fungal; D009687:Nuclear Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D011233:Precipitin Tests; D017434:Protein Structure, Tertiary; D018085:Regulon; D012097:Repressor Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D012441:Saccharomyces cerevisiae / Q000235:genetics* / Q000254:growth & development; D029701:Saccharomyces cerevisiae Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D017384:Sequence Deletion; D016385:TATA Box; D014157:Transcription Factors / Q000235:genetics / Q000378:metabolism
chemical_list: C065455:CYC8 protein, S cerevisiae; D002843:Chromatin; D004268:DNA-Binding Proteins; D005656:Fungal Proteins; C112006:MOT3 protein, S cerevisiae; D009687:Nuclear Proteins; C083348:ROX1 protein, S cerevisiae; D012097:Repressor Proteins; D029701:Saccharomyces cerevisiae Proteins; C066502:TUP1 protein, S cerevisiae; D014157:Transcription Factors