The Cyc8-Tup1 complex inhibits transcription primarily by masking the activation domain of the recruiting protein.
ABSTRACT: 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: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:Cellulase production in filamentous fungus Trichoderma reesei is highly responsive to various environmental cues involving multiple positive and negative regulators. XYR1 (Xylanase regulator 1) has been identified as the key transcriptional activator of cellulase gene expression in T. reesei. However, the precise mechanism by which XYR1 achieves transcriptional activation of cellulase genes is still not fully understood. Here, we identified the TrCYC8/TUP1 complex as a novel coactivator for XYR1 in T. reesei. CYC8/TUP1 is the first identified transcriptional corepressor complex mediating repression of diverse genes in Saccharomyces cerevisiae. Knockdown of Trcyc8 or Trtup1 resulted in markedly impaired cellulase gene expression in T. reesei. We found that TrCYC8/TUP1 was recruited to cellulase gene promoters upon cellulose induction and this recruitment is dependent on XYR1. We further observed that repressed Trtup1 or Trcyc8 expression caused a strong defect in XYR1 occupancy and loss of histone H4 at cellulase gene promoters. The defects in XYR1 binding and transcriptional activation of target genes in Trtup1 or Trcyc8 repressed cells could not be overcome by XYR1 overexpression. Our results reveal a novel coactivator function for TrCYC8/TUP1 at the level of activator binding, and suggest a mechanism in which interdependent recruitment of XYR1 and TrCYC8/TUP1 to cellulase gene promoters represents an important regulatory circuit in ensuring the induced cellulase gene expression. These findings thus contribute to unveiling the intricate regulatory mechanism underlying XYR1-mediated cellulase gene activation and also provide an important clue that will help further improve cellulase production by T. reesei.
Project description:Saccharomyces cerevisiae normally cannot assimilate mannitol, a promising brown macroalgal carbon source for bioethanol production. The molecular basis of this inability remains unknown. We found that cells capable of assimilating mannitol arose spontaneously from wild-type S. cerevisiae during prolonged culture in mannitol-containing medium. Based on microarray data, complementation analysis, and cell growth data, we demonstrated that acquisition of mannitol-assimilating ability was due to spontaneous mutations in the genes encoding Tup1 or Cyc8, which constitute a general corepressor complex that regulates many kinds of genes. We also showed that an S. cerevisiae strain carrying a mutant allele of CYC8 exhibited superior salt tolerance relative to other ethanologenic microorganisms; this characteristic would be highly beneficial for the production of bioethanol from marine biomass. Thus, we succeeded in conferring the ability to assimilate mannitol on S. cerevisiae through dysfunction of Tup1-Cyc8, facilitating production of ethanol from mannitol.
Project description:Environmental stressors can severely perturb cellular homeostasis and compromise viability. To cope with environmental stressors, eukaryotes have developed distinct signaling programs that allow for adaptation during different stress conditions. These programs often require a host of post-translational modifications that alter proteins to elicit appropriate cellular responses. One crucial protein modifier during stress is the small ubiquitin-like modifier SUMO. In many cases, however, the functions of stress dependent protein SUMOylation remain unclear. Previously, we showed that the conserved Saccharomyces cerevisiae Cyc8-Tup1 transcriptional corepressor complex undergoes transient hyperosmotic stress-induced SUMOylation and inclusion formation, which are important for appropriate regulation of hyperosmotic-stress genes. Here, we show the osmostress-responsive MAP kinase Hog1 regulates Cyc8 SUMOylation and inclusion formation via its role in the transcriptional activation of glycerol biosynthesis genes. Mutations that ablate Cyc8 SUMOylation can partially rescue the osmosensitivity of hog1? cells, and this is facilitated by inappropriate derepression of glycerol-biosynthesis genes. Furthermore, cells specifically unable to synthesize the osmolyte glycerol cause transient Cyc8 SUMOylation and inclusions to persist, indicating a regulatory role for glycerol to reestablish the basal state of Cyc8 following adaptation to hyperosmotic stress. These observations unveil a novel intersection between phosphorylation and SUMOylation networks, which are critical for shifting gene expression and metabolic programs during stress adaptation.
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: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 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:Saccharomyces cerevisiae normally cannot assimilate mannitol, a promising brown macroalgal carbon source for bioethanol production. To date, the molecular mechanisms underlying this inability remain unknown. Here, we found that cells acquiring mannitol-assimilating ability appeared from wild-type S. cerevisiae strain during prolonged culture in mannitol medium. Our microarray analysis revealed that genes for putative mannitol dehydrogenase and hexose transporters were up-regulated in cells acquiring mannitol-assimilating ability. Take account of our other results including complementation analysis and cell growth data, we demonstrated that this acquisition of mannitol-assimilating ability was due to the spontaneous mutation in the gene encoding Tup1 or Cyc8. Tup1-Cyc8 is the general corepressor complex involved in the repression of many kinds of genes. Thus, it is suggested that the inability of wild-type S. cerevisiae to assimilate mannitol can be attributed to the transcriptional repression of a set of genes involved in mannitol utilization by Tup1-Cyc8 corepressor. In other words, Tup1-Cyc8 is a key regulator of mannitol metabolism in S. cerevisiae. We also showed that S. cerevisiae strain which carries mutant allele of TUP1 or CYC8 produced ethanol from mannitol efficiently. Especially, strain carrying mutant allele of CYC8 showed high tolerance to salt, which is superior to other ethanologenic microorganisms. This characteristic is highly beneficial to produce bioethanol from marine biomass. Taken together, Tup1-Cyc8 can be an ideal target to develop a yeast-algal bioethanol production system. To figure out how Mtl+ strains (cells acquiring ability to grow in mannitol medium) had acquired the ability to assimilate mannitol, we performed genome-wide analysis by using Nimblegen microarrays. Yeast Saccharomyces cerevisiae cells (wild-type BY4742 strain and two Mtl+ strains, MK3619 and MK3683) were grown at 30°C to the logarithmic phase in SC or SM media. Total RNA was purified and the 4 RNA samples (BY4742 cells in SC as control, MK3619 cells in SM, MK3683 cells in both SC and SM) were analyzed with Nimblegen microarrays.
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.
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: Gene expression in wild-type and tup1(S649F) mutants of a and a mating types was determined by isolation of RNA from logarithmically growing S. cerevisiae cells, followed by Illumina paired-end sequencing. Three replicate RNA samples are included for each strain.