Architectural Mediator subunits are differentially essential for global transcription in yeast (RNA-seq/4tU-seq Med14-Med17-doubleAID)
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ABSTRACT:
PROVIDER: PRJNA610077 | ENA |
REPOSITORIES: ENA
Dataset's files
| Action | DRS | |||
|---|---|---|---|---|
| SRR11232802.fastq.gz | Fastqsanger.gz | |||
| SRR11232803.fastq.gz | Fastqsanger.gz | |||
| SRR11232804.fastq.gz | Fastqsanger.gz | |||
| SRR11232805.fastq.gz | Fastqsanger.gz | |||
| SRR11232806.fastq.gz | Fastqsanger.gz |
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Project description:This project aims to understand the function of different mediator subunits (Med14 and Med17) in transcription in S. cerevisiae. Thus we analyzed total and newly synthesized RNA levels in conditional Med14 and Med17 depletion strains. For this purpose we used an auxin inducible degron system system to specifically induce the degradation Mediator subunits by addition of auxin into the culture medium. We measured transcript levels after 30 min auxin or DMSO (control) treatment induction of degradation of Med14 or Med17 by the addition of auxin (30 min) or with the carrier DMSO (control) followed by a 6 min labelling of newly synthesized RNA with 4-thiouracil (4tU). In parallel wiltype S.pombe cells were labelled with 4tU for 6min during midlog growth phase. Prior to RNA extraction (Ribopure Yeast Kit, Ambion) our labelled S.cerevisiae cells of interest were mixed with labelled S.pombe cells as a spike to allow normalization against an exogenous reference (ratio 3 S .cerevisiae for 1 S.pombe cell. From the total RNA we purified the 4tU labelled fraction of RNAs containing 4tU. Both the total RNA and the purified newly synthesized RNAs will then be subject to RNA-sequencing after ribosomal RNA depletion. In summary, we will provide 16 samples for this project containing RNA from both S.cerevisiae and S.pombe. The sample read counts were normalized with size factors computed by the median-of-ratios method proposed by Anders and Huber (Anders et al. 2010) on S. pombe genes to make these counts comparable between samples.
2020-03-04 | GSE146300 | GEO
Project description:This project aims to understand the function of Mediator and the interplay in transcription in S. cerevisiae. Thus we analyzed total and newly synthesized RNA levels in a conditional Med14 and Med17 depletion strain. For this purpose we used an auxin inducible degron system system to specifically induce the degradation Med14 and Med17 by addition of auxin into the culture medium. We measured transcript levels after 30 min auxin or DMSO (control) treatment induction of degradation of Med14 and Med17 by the addition of auxin (30 min) or with the carrier DMSO (control) followed by a 6 min labelling of newly synthesized RNA with 4-thiouracil (4tU). In parallel wiltype S.pombe cells were labelled with 4tU for 6min during midlog growth phase. Prior to RNA extraction (Ribopure Yeast Kit, Ambion) our labelled S.cerevisiae cells of interest were mixed with labelled S.pombe cells as a spike to allow normalization against an exogenous reference (ratio 3 S .cerevisiae for 1 S.pombe cell. From the total RNA we purified the 4tU labelled fraction of RNAs containing 4tU. Both the total RNA and the purified newly synthesized RNAs will then be subject to RNA-sequencing after ribosomal RNA depletion. In summary, we will provide 8 samples for this project containing RNA from both S.cerevisiae and S.pombe. The sample read counts were normalized with size factors computed by the median-of-ratios method proposed by Anders and Huber (Anders et al. 2010) on S. pombe genes to make these counts comparable between samples.
2020-03-04 | GSE146321 | GEO
Project description:Architectural Mediator subunits are differentially essential for global transcription in yeast (RNA-seq/4tU-seq Med14-AID, Med17-AID)
| PRJNA610054 | ENA
Project description:Architectural Mediator subunits are differentially essential for global transcription in yeast (RNA-seq/4tU-seq)
| PRJNA610215 | ENA
Project description:Architectural Mediator subunits are differentially essential for global transcription in yeast (RNA-seq/4tU-seq Sua7-AID)
| PRJNA610076 | ENA
Project description:Mediator is a modular complex required for RNA polymerase II transcription in vivo. We show that Mediator destabilization via depletion of the main complex scaffold Med14 or the head module scaffold Med17 is lethal and results in global transcriptional downregulation, though Med17 destruction has a markedly greater negative effect. Depletion of Med14 or Med17 impairs pre-initiation complex (PIC) formation and/or stability to similar degrees, suggesting that the differential transcriptional effects of head module destabilization and overall dissociation of the complex are not due to different extents of defective PIC formation. Co-depletion of Med14 and Med17 reduced transcription and TFIIB promoter occupancy to similar degrees as Med17 loss alone, suggesting that the independent head module can weakly stimulate transcription in vivo, though not at a level that maintains viability. Collectively, our data suggest that the head is the major functional module of Mediator with respect to PIC function in vivo.
2020-03-05 | GSE145494 | GEO
Project description:This project aims to understand the function of general transcription factor TFIIB in transcription in S. cerevisiae. Thus, we analyzed total and newly synthesized RNA levels in a conditional Sua7 (TFIIB) depletion strain. For this purpose we used an auxin inducible degron system system to specifically induce the degradation Sua7 by addition of auxin into the culture medium. We measured transcript levels after 30 min auxin or DMSO (control) treatment induction of degradation of Sua7 by the addition of auxin (30 min) or with the carrier DMSO (control) followed by a 6 min labelling of newly synthesized RNA with 4-thiouracil (4tU). In parallel wiltype S.pombe cells were labelled with 4tU for 6min during midlog growth phase. Prior to RNA extraction (Ribopure Yeast Kit, Ambion) our labelled S.cerevisiae cells of interest were mixed with labelled S.pombe cells as a spike to allow normalization against an exogenous reference (ratio 3 S .cerevisiae for 1 S.pombe cell. From the total RNA we purified the 4tU labelled fraction of RNAs containing 4tU. Both the total RNA and the purified newly synthesized RNAs will then be subject to RNA-sequencing after ribosomal RNA depletion. In summary, we will provide 8 samples for this project containing RNA from both S.cerevisiae and S.pombe. The sample read counts were normalized with size factors computed by the median-of-ratios method proposed by Anders and Huber (Anders et al. 2010) on S. pombe genes to make these counts comparable between samples.
2020-03-04 | GSE146318 | GEO
Project description:Transcriptional profiling of 2.25 dpf (days post fertilzation) zebrafish embryos comparing wildtype (control) to med14 (mediator complex subunit 14) mutants. Goal was to determine the extent to which transcription was affected in med14 mutants by analyzing global gene expression. Background: Mediator is an evolutionarily conserved multi-subunit complex that physically links the RNA polymerase II transcriptional machinery to enhancer bound regulatory factors. While classically described as a core component of the transcriptional machinery, recent evidence suggests that Mediator may have context-dependent roles. In particular, a requirement for Mediator in the maintenance of embryonic and induced pluripotent stem cells has been noted. However, the in vivo consequences of loss of function of many Mediator subunits are unknown. Results: We identified med14, which encodes a Mediator subunit, as the gene affected in the zebrafish logelei (log) mutant. log mutants displayed a morphological arrest by 2 days of embryonic development. Surprisingly, microarray analysis showed that transcription was not broadly affected in log mutants. Transplanted log cells placed into a wild-type environment were able to survive into adulthood, suggesting a lack of global transcriptional defects in Med14-deficient cells. RNAi knockdown of the single med14 ortholog in planarians demonstrated that med14 was required for adult stem cell maintenance and regeneration. A broader assay of Mediator subunits revealed that many were required for stem cell maintenance in planarians. Multiple stem/progenitor cell populations were observed to be reduced or absent in zebrafish med14 mutant embryos, with embryonic fin regeneration defects also evident. Conclusions: Taken together, our results show a critical, evolutionarily conserved, function for Med14 (and perhaps Mediator in general) in evolutionarily conserved in vivo stem cell maintenance and regeneration, distinct from a ubiquitous role in transcription.
2017-02-09 | GSE58042 | GEO
Project description:Architectural Mediator subunits are differentially essential for global transcription in yeast (ChIP-seq)
| PRJNA607312 | ENA
Project description:Warm ambient temperatures do not trigger stress responses in plants but stimulate the growth of specific organs. The basic helix-loop-helix transcription factor PHYTOCHROME-INTERACTING FACTOR 4 (PIF4) plays a central role in regulating thermomorphogenetic hypocotyl elongation in various plant species, including the model dicotyledonous plant Arabidopsis thaliana. Although it is well known that PIF4 promotes plant thermosensory growth by activating key genes involved in the biosynthesis and signaling of the phytohormone auxin, the detailed molecular mechanism of such transcriptional activation is still not clear. Our previous studies demonstrated that HEMERA (HMR), a transcription co-activator of PIF4, promotes the thermo-induced expression of PIF4 target genes through the nine-amino-acid transactivation domain (9aaTAD) in its carboxy-terminus. In this report, we investigate the role of the Mediator complex in the PIF4/HMR-mediated thermoresponsive gene expression. Through the characterization of various mutants of the Mediator complex, a tail subunit named MED14 is identified as an essential factor for thermomorphogenetic hypocotyl growth. MED14 is required for the thermal induction of key PIF4 target genes but has a marginal effect on the levels of PIF4 and HMR. Further transcriptomic analyses confirm that the expression of numerous PIF4/HMR-dependent, auxin-related genes requires MED14 at a warm temperature. Moreover, PIF4 and HMR physically interact with MED14 and both are indispensable for the association of MED14 with the promoters of these thermoresponsive genes. Together, these results unveil an important thermomorphogenetic mechanism, in which PIF4 and its coactivator HMR recruit the Mediator complex to activate auxin-related growth-promoting genes when plants sense moderate increases in ambient temperature.
2022-09-26 | GSE196969 | GEO