Project description:The budding yeast transcriptional corepressor Tup1-Ssn6 is a model for studying similar repressosome complexes in multicellular eukaryotes. Tup1-Ssn6 does not bind DNA directly, but is directed to individual promoters by one or more DNA-binding proteins, referred to as Tup1 recruiters. We determined the genomic distribution of Tup1 and Ssn6 by ChIP-chip and found that most loci bound by Tup1-Ssn6 could not be explained by co-occupancy with a known recruiting cofactor. Furthermore, we found that individual deletions of known Tup1 recruiters did not significantly alter Tup1 binding profile. These two observations suggest that Tup1 recruitment typically depends on multiple recruiting cofactors, and that new Tup1 recruiting proteins remain to be discovered. To identify new recruiting proteins we computationally screened for factors with binding patterns similar to the observed Tup1-Ssn6 genomic distribution. Four top candidates, Cin5, Skn7, Phd1, and Yap6, all known to be associated with stress response gene regulation, were experimentally confirmed to physically interact with Tup1 and/or Ssn6. Incorporating these new cofactors with previously characterized cofactors now accounts for the majority of Tup1 binding across the genome, and expands our understanding of the mechanism by which Tup1-Ssn6 is directed to its targets.
Project description:The budding yeast transcriptional corepressor Tup1-Ssn6 is a model for studying similar repressosome complexes in multicellular eukaryotes. Tup1-Ssn6 does not bind DNA directly, but is directed to individual promoters by one or more DNA-binding proteins, referred to as Tup1 recruiters. We determined the genomic distribution of Tup1 and Ssn6 by ChIP-chip and found that most loci bound by Tup1-Ssn6 could not be explained by co-occupancy with a known recruiting cofactor. Furthermore, we found that individual deletions of known Tup1 recruiters did not significantly alter Tup1 binding profile. These two observations suggest that Tup1 recruitment typically depends on multiple recruiting cofactors, and that new Tup1 recruiting proteins remain to be discovered. To identify new recruiting proteins we computationally screened for factors with binding patterns similar to the observed Tup1-Ssn6 genomic distribution. Four top candidates, Cin5, Skn7, Phd1, and Yap6, all known to be associated with stress response gene regulation, were experimentally confirmed to physically interact with Tup1 and/or Ssn6. Incorporating these new cofactors with previously characterized cofactors now accounts for the majority of Tup1 binding across the genome, and expands our understanding of the mechanism by which Tup1-Ssn6 is directed to its targets. ChIP-chip
Project description:The yeast 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 Ssn6-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 SSN6 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 SSN6 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 Ssn6 and Tup1 preferentially bind TATA-containing promoters, which are also enriched in genes derepressed upon loss of SSN6 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 Ssn6-Tup1 corepressor, and that releasing the P nucleosome contributes to gene activation.
Project description:The yeast 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 Ssn6-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 SSN6 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 SSN6 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 Ssn6 and Tup1 preferentially bind TATA-containing promoters, which are also enriched in genes derepressed upon loss of SSN6 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 Ssn6-Tup1 corepressor, and that releasing the P nucleosome contributes to gene activation.
Project description:The yeast 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 Ssn6-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 SSN6 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 SSN6 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 Ssn6 and Tup1 preferentially bind TATA-containing promoters, which are also enriched in genes derepressed upon loss of SSN6 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 Ssn6-Tup1 corepressor, and that releasing the P nucleosome contributes to gene activation. nucleosomes were prepared from isogenic wild type (BY4742), ssn6 KO and tup1 KO cells after varying degrees of micrococcal nuclease (MNase) digestion, followed by isolation of mononucleosomal DNA and sequencing. Three replicates of each strain (9 samples) were subjected to Illumina sequencing.
Project description:The yeast 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 Ssn6-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 SSN6 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 SSN6 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 Ssn6 and Tup1 preferentially bind TATA-containing promoters, which are also enriched in genes derepressed upon loss of SSN6 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 Ssn6-Tup1 corepressor, and that releasing the P nucleosome contributes to gene activation. Genome-wide expression profiling Yeast gene expression in three cell type, Each cell type is tested in duplicate.
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.
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.