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A SAGA-Independent Function of SPT3 Mediates Transcriptional Deregulation in a Mutant of the Ccr4-Not Complex

ABSTRACT: The conserved multi-subunit Ccr4-Not complex regulates gene expression in diverse ways. In this work, we characterize the suppression of temperature sensitivity associated with a mutation in the gene encoding the scaffold subunit of the Ccr4-Not complex, NOT1, by the deletion of SPT3. We determine that the deletion of SPT3, but not the deletion of genes encoding other subunits of the SAGA complex, globally suppresses transcriptional defects of not1-2. We find that transcriptional activation in not1-2 is associated with increased binding of TFIID and SAGA at promoters of upregulated genes, and this is suppressed by the deletion of SPT3. Interestingly, Spt3p-dependent activation of transcription occurs in not1-2 even if the SAGA complex is disrupted by the deletion of SPT7 that encodes a subunit of SAGA required for its integrity. Consistent with a SAGA-independent function of Spt3p, the deletion of SPT3 displays synthetic phenotypes when combined with a deletion of SPT7. Taken together, our results provide a new view of the Spt3 protein by identifying a SAGA-independent function of this protein that is functionally linked to the Ccr4-Not complex. Keywords: genetic modification

ORGANISM(S): Saccharomyces cerevisiae

PROVIDER: GSE9432 | GEO | 2007/10/26

SECONDARY ACCESSION(S): PRJNA103181

REPOSITORIES: GEO

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Transcription profiling of S. cerevisiae wild type reference strain, two simple mutants (not1-2 and spt3) and one double mutant (not1-2 spt3) reveals a SAGA-Independent function of SPT3 mediates transcriptional deregulation in a mutant of the Ccr4-Not Complex

Project description:The conserved multi-subunit Ccr4-Not complex regulates gene expression in diverse ways. In this work, we characterize the suppression of temperature sensitivity associated with a mutation in the gene encoding the scaffold subunit of the Ccr4-Not complex, NOT1, by the deletion of SPT3. We determine that the deletion of SPT3, but not the deletion of genes encoding other subunits of the SAGA complex, globally suppresses transcriptional defects of not1-2. We find that transcriptional activation in not1-2 is associated with increased binding of TFIID and SAGA at promoters of upregulated genes, and this is suppressed by the deletion of SPT3. Interestingly, Spt3p-dependent activation of transcription occurs in not1-2 even if the SAGA complex is disrupted by the deletion of SPT7 that encodes a subunit of SAGA required for its integrity. Consistent with a SAGA-independent function of Spt3p, the deletion of SPT3 displays synthetic phenotypes when combined with a deletion of SPT7. Taken together, our results provide a new view of the Spt3 protein by identifying a SAGA-independent function of this protein that is functionally linked to the Ccr4-Not complex. Experiment Overall Design: 4 strains were analyzed: the wild type reference strain, two simple mutants (not1-2 and spt3) and one double mutant (not1-2 spt3). All strains were in duplicate.

2008-06-16 | E-GEOD-9432 | biostudies-arrayexpress

The CCR4-NOT complex component NOT1 regulates RNA-directed DNA methylation and transcriptional silencing by facilitating Pol IV-dependent siRNA production [Bisulfite-seq]

Project description:Small interfering RNAs (siRNAs) are required for establishment and maintenance of DNA methylation through RNA-directed DNA methylation (RdDM) pathway in plants. While the siRNA biogenesis is well known, relatively little is known about how the process is regulated. By a forward genetic screen in Arabidopsis thaliana, we identified a mutant defective in NOT1 and demonstrated that NOT1 is required for transcriptional silencing at RdDM target genomic loci. Furthermore, we revealed that NOT1 is required for Pol IV-dependent siRNA accumulation and DNA methylation at a subset of RdDM target loci. By immunoprecipitation combined with mass spectrometry, we showed that NOT1 is a constituent of a multi-subunit CCR4-NOT deadenylase complex. We demonstrated that the CCR4-NOT components can function as a whole to mediate chromatin silencing. Our work thus establishes that the CCR4-NOT complex regulates the production of Pol IV-dependent siRNAs and thereby facilitates DNA methylation and transcriptional silencing in Arabidopsis.

2020-05-15 | GSE148428 | GEO

The CCR4-NOT complex component NOT1 regulates RNA-directed DNA methylation and transcriptional silencing by facilitating Pol IV-dependent siRNA production [small RNA-seq]

2020-05-15 | GSE148429 | GEO

Identification of acetylation-regulated interactors of the human CCR4-NOT complex

Project description:The evolutionarily conserved CCR4-NOT complex acts as a central player in the control of mRNA turnover and mediates accelerated mRNA degradation upon histone deacetylase (HDAC) inhibition. Here, we explored acetylation-induced changes in the composition of the CCR4-NOT complex by purification of the endogenously tagged scaffold subunit NOT1 and identified ring finger protein 219 (RNF219) as an acetylation-regulated cofactor. We provide evidence that RNF219 is an active RING-type E3 ubiquitin ligase which stably associates with the CCR4-NOT complex via the NOT9 module through a short linear motif located in the C-terminal low-complexity region of RNF219. Using a reconstituted six-subunit human CCR4-NOT complex, we demonstrate that RNF219 acts as an inhibitor of deadenylation. Accordingly, our transcriptome-wide mRNA half-life measurements reveal that RNF219 attenuates global mRNA turnover in cells, independently of its E3 ligase activity. Our results establish RNF219 as a negative regulator of CCR4-NOT-mediated mRNA deadenylation, whose loss upon HDAC inhibition contributes to accelerated mRNA turnover.

2021-12-15 | PXD027237 | Pride

Specific Roles for the Ccr4-Not Complex Subunits in Expression of the Genome

2008-12-10 | GSE10521 | GEO

Next Generation Sequencing Analysis of Maize Wild Type (WT) and thk1 Mutants Transcriptomes

Project description:The thk1 mutant, causing multiple aleurone layers, disrupts the NOT1 scaffolding subunit of the CCR4-NOT regulatory complex and dysregulates genes involved in cell division, signaling, differentiation and metabolism.

2020-07-29 | GSE155296 | GEO

Specific Roles for the Ccr4-Not Complex Subunits in Expression of the Genome

Project description:These Affymetrix data were used to determine the role of each non-essential subunit of the conserved Ccr4-Not complex in the control of gene expression in the yeast S. cerevisiae. The study was performed with cells growing exponentially in high glucose and with cells grown to glucose depletion. Specific patterns of gene de-regulation were observed upon deletion of any given subunit, revealing the specificity of each subunit’s function. Consistently, the purification of the Ccr4-Not complex through Caf40p by tandem affinity purification from wild-type cells or cells lacking individual subunits of the Ccr4-Not complex revealed that each subunit had a particular impact on complex integrity. Furthermore, the micro-arrays revealed that the role of each subunit was specific to the growth conditions. From the study of only two different growth conditions, revealing an impact of the Ccr4-Not complex on more than 85% of all studied genes, we can infer that the Ccr4-Not complex is important for expression of most of the yeast genome. Keywords: genetic modification, stress response Wild-type and not2?, not3?, not4?, not5?, caf1?, caf130? and ccr4? null strains were grown in glucose rich medium to exponential phase and collected. Wild-type and not2?, not4?, caf1?, caf130? and ccr4? null strains were also grown in glucose rich medium to glucose depletion and collected 30 min after glucose depletion. Each sample was prepared twice independently. One array from the caf1? strain grown to glucose depletion, and one from the not5? and not4? strains growing exponentially were found to be problematic and discarded. A not5? sample was not prepared after glucose depletion, because it flocculates in that condition.

2008-12-10 | E-GEOD-10521 | biostudies-arrayexpress

Acetylation-dependent Multimerization of SAGA Regulates Gene Transcription

Project description:In Saccharomyces cerevisiae, the SAGA complex regulates its own activity by undergoing multimerization. This multimerization is triggered by SAGA autoacetylation at three sites on its Ada3 subunit, allowing recognition of this acetylation by the bromodomain of the Gcn5/Spt7 SAGA subunit. Once multimerized, SAGA is capable of cooperatively acetylating chromatin, and an inability to autoacetylate Ada3 leads to transcriptional and phenotypic defects in a wide range of stress-activated genes. The SAGA multimerization increased significantly in media with Sucorse as the only carbon resource than that with Glucose. In this study, the high-throughput sequence of wild type (WT) strain, Ada3 acetylation mutant (Ada3-3KR), Gcn5 bromodomain mutant (Gcn5m) and Spt7 bromodomain mutant (Spt7m) indicate a new function for Ada3 acetylation, show which genes transcription can be regulated by SAGA multimers.

2021-11-29 | GSE161887 | GEO

RNAi knock-down of CAF1-CCR4-NOT1 deadenylase complex or AGO1 in D. melanogaster S2 cells

Project description:miRNAs silence gene expression by repressing translation and/or by promoting mRNA decay. AGO1 is a key protein required for miRNA-mediated gene silencing. In animal cells, mRNA degradation of partially complementary miRNA targets occurs via deadenylation by the CAF1-CCR4-NOT1 deadenylase complex, followed by decapping and subsequent exonucleolytic digestion. To determine how generally miRNAs trigger deadenylation, we compared mRNA expression profiles in D. melanogaster cells depleted of AGO1, CAF1 or NOT1. We show that approximately 45% of AGO1-targets are regulated by both CAF1 and NOT1, indicating deadenylation is a widespread effect of miRNA regulation.<br><br>We employed RNA interference using long double-stranded RNAs to deplete cultured S2 cells of AGO1 (CG6671, 2 independent samples), CAF1 (CG5684, 2 independent samples), or NOT1 (CG1884, 3 independent samples). Further, we used Affymetrix oligonucleotide microarrays to analyze expression profiles in these samples. We included the following controls: mock RNAi treatment and GFP dsRNA treatment (3 and 2 independent samples, respectively).<br>

2008-12-03 | E-MEXP-1713 | biostudies-arrayexpress

The CCR4-Not complex is a tumor suppressor in Drosophila melanogaster eye cancer models

Project description:The CNOT3 protein is a subunit of the CCR4-Not complex, a cytoplasmic protein complex mediating mRNA deadenylation as the first step in mRNA degradation. We recently identified CNOT3 loss-of-function mutations in patients with T-cell acute lymphoblastic leukemia (T-ALL). However, it is not clear yet how loss of this gene is involved in cancer development. Here we use different Drosophila melanogaster eye cancer models to study the potential tumor suppressor function of Not3, the CNOT3 orthologue, as well as other members of the CCR4-NOT complex. We show that not only Not3 but also the structural components Not1 and Not2, as well as the deadenylases Twin and Pop2 all behave as tumor suppressor genes. Moreover, RNA-sequencing analyses revealed that Not3 loss leads to increased expression levels of genes involved in DNA replication and ribosome biogenesis pathways, and that CycB is a Not3 target gene in our cancer models. Furthermore, we were able to confirm those results on human T-ALL cell lines, pointing out the conserved function of Not3 in both model systems and in tumor development. Together, our data establish a critical role for Not3 and the entire CCR4-NOT complex as a tumor suppressor.

2017-09-15 | GSE103872 | GEO

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