Functional dissection of the global repressor Tup1 in yeast: dominant role of the C-terminal repression domain.
ABSTRACT: In the yeast Saccharomyces cerevisiae, Tup1, in association with Cyc8 (Ssn6), functions as a general repressor of transcription. Tup1 and Cyc8 are required for repression of diverse families of genes coordinately controlled by glucose repression, mating type, and other mechanisms. This repression is mediated by recruitment of the Cyc8-Tup1 complex to target promoters by sequence-specific DNA-binding proteins. We created a library of XhoI linker insertions and internal in-frame deletion mutations within the TUP1 coding region. Insertion mutations outside of the WD domains were wild type, while insertions within the WD domains induced mutant phenotypes with differential effects on the target genes SUC2, MFA2, RNR2, and HEM13. Deletion mutations confirmed previous findings of two separate repression domains in the N and C termini. The cumulative data suggest that the C-terminal repression domain, located near the first WD repeat, plays the dominant role in repression. Although the N-terminal repression domain is sufficient for partial repression, deletion of this region does not compromise repression. Surprisingly, deletion of the majority of the histone-binding domain of Tup1 also does not significantly reduce repression. The N-terminal region containing potential alpha-helical coiled coils is required for Tup1 oligomerization and association with Cyc8. Association with Cyc8 is required for repression of SUC2, HEM13, and RNR2 but not MFA2 and STE2.
Project description:The TUP1 and CYC8 (= SSN6) genes of Saccharomyces cerevisiae play a major role in glucose repression. Mutations in either TUP1 or CYC8 eliminate or reduce glucose repression of many repressible genes and induce other phenotypes, including flocculence, failure to sporulate, and sterility of MAT alpha cells. The TUP1 gene was isolated in a screen for genes that regulate mating type (V.L. MacKay, Methods Enzymol. 101:325-343, 1983). We found that a 3.5-kb restriction fragment was sufficient for complete complementation of tup1-100. The gene was further localized by insertional mutagenesis and RNA mapping. Sequence analysis of 2.9 kb of DNA including TUP1 revealed only one long open reading frame which predicts a protein of molecular weight 78,221. The predicted protein is rich in serine, threonine, and glutamine. In the carboxyl region there are six repeats of a pattern of about 43 amino acids. This same pattern of conserved residues is seen in the beta subunit of transducin and the yeast CDC4 gene product. Insertion and deletion mutants are viable, with the same range of phenotypes as for point mutants. Deletions of the 3' end of the coding region produced the same mutant phenotypes as did total deletions, suggesting that the C terminus is critical for TUP1 function. Strains with deletions in both the CYC8 and TUP1 genes are viable, with phenotypes similar to those of strains with a single deletion. A deletion mutation of TUP1 was able to suppress the snf1 mutation block on expression of the SUC2 gene encoding invertase.
Project description:The yeast Xanthophyllomyces dendrorhous produces carotenoids of commercial interest, including astaxanthin and ?-carotene. Although carotenogenesis in this yeast and the expression profiles of the genes controlling this pathway are known, the mechanisms regulating this process remain poorly understood. Several studies have demonstrated that glucose represses carotenogenesis in X. dendrorhous, suggesting that this pathway could be regulated by catabolic repression. Catabolic repression is a highly conserved regulatory mechanism in eukaryotes and has been widely studied in Saccharomyces cerevisiae. Glucose-dependent repression is mainly observed at the transcriptional level and depends on the DNA-binding regulator Mig1, which recruits the co-repressor complex Cyc8-Tup1, which then represses the expression of target genes. In this work, we studied the regulation of carotenogenesis by catabolic repression in X. dendrorhous, focusing on the role of the co-repressor complex Cyc8-Tup1.The X. dendrorhous CYC8 and TUP1 genes were identified, and their functions were demonstrated by heterologous complementation in S. cerevisiae. In addition, cyc8 - and tup1 - mutant strains of X. dendrorhous were obtained, and both mutations were shown to prevent the glucose-dependent repression of carotenogenesis in X. dendrorhous, increasing the carotenoid production in both mutant strains. Furthermore, the effects of glucose on the transcript levels of genes involved in carotenogenesis differed between the mutant strains and wild-type X. dendrorhous, particularly for genes involved in the synthesis of carotenoid precursors, such as HMGR, idi and FPS. Additionally, transcriptomic analyses showed that cyc8 - and tup1 - mutations affected the expression of over 250 genes in X. dendrorhous.The CYC8 and TUP1 genes are functional in X. dendrorhous, and their gene products are involved in catabolic repression and carotenogenesis regulation. This study presents the first report involving the participation of Cyc8 and Tup1 in carotenogenesis regulation in yeast.
Project description:In yeast, global genome nucleotide-excision repair (GG-NER) requires a protein complex containing Rad7 and Rad16. Rad16 is a member of the switch/sucrose nonfermentable superfamily, and it is presumed that chromatin remodelling is its primary function during repair. We show that RAD16 is required for ultraviolet-dependent hyperacetylation of histone H3 (Lys 9 and Lys 14) at the MFA2 promoter and throughout the genome. The yeast repressor complex Ssn6-Tup1 represses many genes including MFA2. TUP1 deletion results in constitutive hyperacetylation of histone H3, nucleosome disruption and derepression of gene transcription in Tup1-regulated genes. GG-NER in the MFA2 promoter proceeds more rapidly in tup1Delta alpha-cells compared with wild type, even when transcription is inhibited. We show that elevated histone H3 acetylation levels in the MFA2 promoter in tup1Delta alpha-cells result in Rad7- and Rad16-independent GG-NER, and that Rad16 mediates the ultraviolet-induced acetylation of histone H3, necessary for efficient GG-NER.
Project description:Intrinsic signals and external cues from the environment drive cell fate decisions. In budding yeast, the decision to enter meiosis is controlled by nutrient and mating-type signals that regulate expression of the master transcription factor for meiotic entry, IME1. How nutrient signals control IME1 expression remains poorly understood. Here, we show that IME1 transcription is regulated by multiple sequence-specific transcription factors (TFs) that mediate association of Tup1-Cyc8 co-repressor to its promoter. We find that at least eight TFs bind the IME1 promoter when nutrients are ample. Remarkably, association of these TFs is highly regulated by different nutrient cues. Mutant cells lacking three TFs (Sok2/Phd1/Yap6) displayed reduced Tup1-Cyc8 association, increased IME1 expression, and earlier onset of meiosis. Our data demonstrate that the promoter of a master regulator is primed for rapid activation while repression by multiple TFs mediating Tup1-Cyc8 recruitment dictates the fate decision to enter meiosis.
Project description:In Saccharomyces cerevisiae expression of the fructose-1,6-bisphosphatase-encoding gene, FBP1, is controlled by glucose through the upstream activating sequences UAS1 and UAS2 and the upstream repressing sequence URS1 in its promoter. We have studied the regulation of the proteins that could bind to these elements. We have investigated the role of the putative transcription factors Cat8 and Sip4 in the formation of specific DNA-protein complexes with UAS1 and UAS2, and in the expression of UAS1-lacZ and UAS2-lacZ. The expression of CAT8-lacZ and SIP4-lacZ has been also measured in mig1, tup1 or hxk2 mutants, partially refractory to catabolite repression. We conclude that there is no strict correlation between Cat8 and Sip4 expression or in vitro formation of DNA-protein complexes and expression of UAS1-lacZ and UAS2-lacZ. The URS1 element binds the regulatory protein Mig1, which blocks transcription by recruiting the proteins Cyc8 and Tup1. The pattern of complexes of URS1 with nuclear extracts was dependent on the carbon source and on Cyc8, but not on Tup1; it was also affected by the protein kinase Snf1 and by the exportin Msn5. The repression caused by URS1 in a fusion gene was dependent on Mig1, Cyc8 and Tup1, and on the carbon source in the medium; in a snf1 strain the repression observed was independent of the carbon source. Expression of Mig1 could occur in the absence of Snf1 and was moderately sensitive to glucose. We present data showing that different elements of the regulatory system controlling FBP1 responded differently to the concentration of glucose in the medium.
Project description:Tup1-Cyc8 (also known as Tup1-Ssn6) is a general transcriptional corepressor. D-Mannitol (mannitol) and D-sorbitol (sorbitol) are the major polyols in nature. Budding yeast Saccharomyces cerevisiae is unable to assimilate mannitol or sorbitol, but acquires the ability to assimilate mannitol due to a spontaneous mutation in TUP1 or CYC8. In this study, we found that spontaneous mutation of TUP1 or CYC8 also permitted assimilation of sorbitol. Some spontaneous nonsense mutations of CYC8 produced a truncated Cyc8 with a C-terminal polyglutamine. The effects were guanidine hydrochloride-sensitive and were dependent on Hsp104, but were complemented by introduction of CYC8, ruling out involvement of a prion. Assimilation of mannitol and sorbitol conferred by other mutations of TUP1 or CYC8 was guanidine hydrochloride-tolerant. It is physiologically reasonable that S. cerevisiae carries this mechanism to acquire the ability to assimilate major polyols in nature.
Project description:The yeast Cyc8 (also known as Ssn6)-Tup1 complex regulates gene expression through a variety of mechanisms, including positioning of nucleosomes over promoters of some target genes to limit accessibility to the transcription machinery. To further define the functions of Cyc8-Tup1 in gene regulation and chromatin remodeling, we performed genome-wide profiling of changes in nucleosome organization and gene expression that occur upon loss of CYC8 or TUP1 and observed extensive nucleosome alterations in both promoters and gene bodies of derepressed genes. Our improved nucleosome profiling and analysis approaches revealed low-occupancy promoter nucleosomes (P nucleosomes) at locations previously defined as nucleosome-free regions. In the absence of CYC8 or TUP1, this P nucleosome is frequently lost, whereas nucleosomes are gained at -1 and +1 positions, accompanying up-regulation of downstream genes. Our analysis of public ChIP-seq data revealed that Cyc8 and Tup1 preferentially bind TATA-containing promoters, which are also enriched in genes derepressed upon loss of CYC8 or TUP1. These results suggest that stabilization of the P nucleosome on TATA-containing promoters may be a central feature of the repressive chromatin architecture created by the Cyc8-Tup1 corepressor, and that releasing the P nucleosome contributes to gene activation.
Project description:The yeast Tup1-Cyc8 corepressor complex is recruited to promoters by DNA-binding repressors, but the mechanisms by which it inhibits expression of genes involved in various stress pathways are poorly understood. Conditional and rapid depletion of Tup1 from the nucleus leads to concurrent nucleosome depletion and histone acetylation, recruitment of coactivators (Swi/Snf, SAGA, and Mediator), and increased transcriptional activity. Conversely, coactivator dissociation occurs rapidly upon rerepression by Cyc8-Tup1, although coactivator association and transcription can be blocked even in the absence of nucleosomes. The coactivators are recruited to the sites where Tup1 was located prior to depletion, indicating that the repressor proteins that recruit Tup1 function as activators in its absence. Last, Cyc8-Tup1 can interact with activation domains in vivo. Thus, Cyc8-Tup1 regulates transcription primarily by masking and inhibiting the transcriptional activation domains of the recruiting proteins, not by acting as a corepressor. We suggest that the corepressor function of Cyc8-Tup1 makes only a modest contribution to expression of target genes, specifically to keep expression levels below the nonactivated state.
Project description:In budding yeast, Tup1 and Ssn6/Cyc8 form a corepressor that regulates a large number of genes. This Tup1-Ssn6 corepressor appears to be conserved from yeast to man. In the pathogenic fungus Candida albicans, Tup1 regulates cellular morphogenesis, phenotypic switching, and metabolism, but the role of Ssn6 remains unclear. We show that there are clear differences in the morphological and invasive phenotypes of C. albicans ssn6 and tup1 mutants. Unlike Tup1, Ssn6 depletion promoted morphological events reminiscent of phenotypic switching rather than filamentous growth. Transcript profiling revealed minimal overlap between the Ssn6 and Tup1 regulons. Hypha-specific genes, which are repressed by Tup1 and Nrg1, were not derepressed in ssn6 cells under the conditions studied. In contrast, the phase specific gene WH11 was derepressed in ssn6 cells, but not in tup1 or nrg1 cells. Hence Ssn6 and Tup1 play distinct roles in C. albicans. Nevertheless, both Ssn6 and Tup1 were required for the Nrg1-mediated repression of an artificial NRE promoter, and lexA-Nrg1 mediated repression in the C. albicans one-hybrid system. These observations are explained in models that are generally consistent with the Tup1-Ssn6 paradigm in budding yeast.
Project description:The Tup1-Cyc8 corepressor complex of Saccharomyces cerevisiae is recruited to promoters by DNA-binding proteins to repress transcription of genes, including the a-specific mating type genes. We report here a tup1(S649F) mutant that displays mating irregularities similar to a tup1 null and an a-predominant growth defect. RNA-Seq and ChIP-Seq were used to analyze gene expression and Tup1 binding changes in mutant vs. wild-type in both a and a cells. Increased Tup1(S649F) binding tended to occur upstream of upregulated genes, whereas locations with decreased binding usually did not show changes in gene expression, suggesting this mutant not only loses corepressor function but also behaves as a coactivator. Based upon studies demonstrating a dual role of Tup1 in both repression and activation, we postulate that the coactivator function of Tup1(S649F) results from diminished interaction with repressor proteins, including a2. We also found that large changes in mating type-specific gene expression between a and a or between mutant and wild-type were not easily explained by the range of Tup1 binding levels within their promoters, as predicted by the classic model of a-specific gene repression by Tup1. Most surprisingly, we observed Tup1 binding upstream of the a-specific gene MFA2 and the a-specific gene MF(ALPHA)1 in cells in which each gene was expressed rather than repressed. These results, combined with identification of additional mating related genes upregulated in the tup1(S649F) a strain, illustrate that the role of Tup1 in distinguishing mating types in yeast appears to be both more comprehensive and more nuanced than previously appreciated. Overall design: Genomic localization of Tup1-V5 and Tup1(S649F)-V5 was determined by chromatin immunoprecipitation from cross-linked S. cerevisiae cells, followed by Illumina paired-end sequencing. Three replicate ChIPs and a single input control are included for each strain.