PIF4/HEMERA-mediated daytime thermosensory growth requires the Mediator subunit MED14
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ABSTRACT:
PROVIDER: PRJNA808090 | ENA |
REPOSITORIES: ENA
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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
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: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:The ring-like cohesin complex plays an essential role in chromosome segregation, organization, and double-strand break repair through its ability to bring two DNA double helices together. Scc2 (NIPBL in humans) together with Scc4 function as the loader of cohesin onto chromosomes. Chromatin adapters such as the RSC complex facilitate localization of the Scc2-Scc4 cohesin loader. Here we identify a broad range of Scc2- chromatin protein interactions that are evolutionarily conserved and reveal a role for one complex, Mediator, in recruitment of the cohesin loader. We identified budding yeast Med14, a subunit of the Mediator complex, as a high copy suppressor of poor growth in Scc2 mutant strains. Physical and genetic interactions between Scc2 and Mediator are functionally substantiated in direct recruitment and cohesion assays. Depletion of Med14 results in defective sister chromatid cohesion and decreased binding of Scc2 at RNA Pol II transcribed genes. Previous work has suggested that Mediator, Nipbl, and cohesin connect enhancers and promoters of active mammalian genes. Our studies suggest an evolutionarily conserved fundamental role for Mediator in direct recruitment of Scc2 to RNA pol II transcribed genes. We identified two mutations in the evolutionarily conserved HEAT domain of SCC2 that result in significantly reduced growth, scc2R787G and scc2G1242V. This experiment uses ChIP Seq to examine global localization of Scc2 in the presence or absence of MED14.
2022-05-25 | GSE201193 | 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:BES1 transcription factor is a master regulator of brassinosteroid signalling. Part of BES1 activity as a transcription factor is brought about upon dimerization with light-responsive PIF4 transcription factor. To analyse BES1-dependent transcriptional cascade, and to which extent BES1 requires PIF4/PIF5 for target gene regulation, we performed microarray analysis of the following phenotypes: Col-0, bes1-D, pif4pif5 and bes1-D;pif4pif5 in dark grown seedlings.
2016-08-05 | GSE85211 | GEO
Project description:BES1 transcription factor is a master regulator of brassinosteroid signalling. Part of BES1 activity as a transcription factor is brought about upon dimerization with light-responsive PIF4 transcription factor. To analyse BES1-dependent transcriptional cascade, and to which extent BES1 requires PIF4/PIF5 for target gene regulation, we performed microarray analysis of the following phenotypes: Col-0, bes1-D, pif4pif5 and bes1-D;pif4pif5 in dark grown seedlings. Col-0, bes1-D, pif4pif5 and bes1-D;pif4pif5 5-days seedlings grown in the dark were used for RNA extraction. 3 independent biological samples were collected for each genoptye.
2016-08-05 | E-GEOD-85211 | biostudies-arrayexpress
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:To investigate the effect of the interaction between the MRG1/2 and PIF4, here we analysed differentially expressed genes compared to the mutant and wild-type by RNA-seq under different temperature. We performed gene expression profiling analysis using data obtained from RNA-seq of WT, mrg1 mrg2, pif4, and mrg1 mrg2 pif4 plants at 22degC and 28degC.
2024-01-04 | GSE213025 | GEO
Project description:The Mediator complex transmits activation signals from DNA bound transcription factors to the core transcription machinery. Genome wide localization studies have demonstrated that Mediator occupancy not only correlates with high levels of transcription, but that the complex also is present at transcriptionally silenced locations. We provide evidence that Mediator localization is guided by an interaction with histone tails, and that this interaction is regulated by their post-translational modifications. A quantitative, high-density genetic interaction map revealed links between Mediator components and factors affecting chromatin structure, especially histone deacetylases. Peptide binding assays demonstrated that pure wild type Mediator forms stable complexes with the tails of Histone H3 and H4. These binding assays also showed Mediator – histone H4 peptide interactions are specifically inhibited by acetylation of the histone H4 lysine 16, a residue critical in transcriptional silencing. Finally, these findings were validated by tiling array analysis, that revealed a broad correlation between Mediator and nucleosome occupancy in vivo, but a negative correlation between Mediator and nucleosomes acetylated at histone H4 lysine 16. Our studies show that chromatin structure and the acetylation state of histones are intimately connected to Mediator localization. Med8-TAP strain ChIPed with IgG beads vs. Input in Saccharomyces cerevisiae
2011-11-22 | E-GEOD-28613 | biostudies-arrayexpress