Repression by the Arabidopsis TOPLESS corepressor requires association with the core mediator complex
- Department of Biology, University of Washington, United States
- Department of Pharmacology, United States
- Howard Hughes Medical Institute, University of Washington, United States
Figures
Figure 1 with 1 supplement
The N-terminal domain of TPL contains two independent repression domains.
(A) Schematic of the ARCSc. The auxin-responsive promoter driving the fluorescent protein Venus carries binding sites for the auxin-responsive transcription factor (ARF). In the absence of auxin, …
Figure 1—figure supplement 1
Helix 1 and the CRA domain (Helix 3-Helix 8) can act independently to repress transcription.
Each color represents two independent time-course flow cytometry experiments of the TPL helices indicated, all fused to IAA3 or IAA3 alone (black line). Every point represents the average …
Figure 2 with 2 supplements
The Helix 8 repression domain of TPL directly interacts with AtMED21 and AtMED10B.
(A–C, E) Cytoplasmic split-ubiquitin system (CytoSUS) assays with candidate interacting proteins. Nub-3xHA is the N-terminal fragment of ubiquitin expressed with no fusion protein and is used as a …
Figure 2—figure supplement 1
The TPL-N terminal domain (TPLN188) interacts with the N-terminus of AtMED21.
(A) Identifying TPL-N terminal domain interactor proteins through yeast two hybrid screening identifies TPL as a problematic bait protein as it may silence the activation despite successful binding …
Figure 2—figure supplement 2
Homology and structure of the MED21 subunit of the Mediator complex.
(A) Protein alignment of selected MED21 homologs from various species. Dr: Drosophila melanogaster; Tr: Takifugu rubripes; Ms: Mus musculus; Gg: Gallus gallus; Hs: Homo sapiens; Sp: Strongylocentrotu…
Figure 3
Identification of critical residues within Helix 8 repression domain.
(A) Sequence and structure of Helix 8 (5NQS). Helix 8 is colored green, and amino acids chosen for mutation are highlighted in light green in both the sequence and the structure. (B) Repression …
Figure 4 with 4 supplements
Repression by TPL requires interaction with the N-terminus of MED21 at promoters.
(A) Model of the proposed interaction between the TPL N-terminus with Mediator, where TPL interaction with Mediator 21 and 10 inhibits the recruitment of Pol II. Proteins in this complex that were …
Figure 4—figure supplement 1
Construction and characterization of the single locus auxin response circuit (SPARC).
(A) Design schematic of the approach utilized to create the SPARC through a Versatile Genetic Assembly System (VEGAS) assembly approach. Each individual transcriptional unit (TU) was checked to …
Figure 4—figure supplement 2
Mediator is detectable at the ARC promoter.
(A) Cartoon schematic of the ARC reporter. The primers flanking the ARF binding site (A) and Venus gene body (B) are highlighted. The B primer set is ~500 bp downstream of the A primer set. The …
Figure 4—figure supplement 3
N-terminal ScMed21 deletions impair auxin-responsive transcriptional activation.
(A) Time-course flow cytometry analysis of SPARCH1-H5 in wild-type and n-terminal ScMed21 deletions with and without auxin. Genotypes are indicated in the colored key inset into the graph. Auxin …
Figure 4—figure supplement 4
Inducible MED21 rescues rapamycin-induced yeast growth defects.
(A) Depletion of nuclear ScMed21 by rapamycin increased cell size even in short time courses, consistent with its essential role in many core pathways. Scatterplots of side scatter area by forward …
Figure 5 with 1 supplement
Multimerization is not required for repression in yeast.
(A) TPL can form a homotetramer via the CRA (red) and LisH (blue) domains. Asterisks indicate mutations that block or diminish these interactions. (B, C) Locations of critical positions in Helix 1 …
Figure 5—figure supplement 1
TPL multimerization requires Helix 8.
(A) Cytoplasmic split-ubiquitin interaction (cytoSUS) assay on serial deletions of TPL. Interaction of bait and prey proteins reconstitutes split ubiquitin, releasing a synthetic transcription …
Figure 6 with 2 supplements
The TPL CRA repression domain behaves similarly in yeast and plants.
(A) Bimolecular fluorescence complementation assay performed in tobacco. Each image is an epi-fluorescent micrograph taken at identical magnification from tobacco epidermal cells at 2 days post …
Figure 6—figure supplement 1
The TPL-MED21 interaction is required for repression in plants.
(A) MED21 and TPL co-immunoprecipitated with AtMED10B from tobacco extracts. Each construct was expressed under the viral 35S promoter, and tissues were harvested after 2 days of injection. MED10B …
Figure 6—figure supplement 2
The essential gene MED21 is required for normal lateral root development in plants.
(A) Identification and characterization of a novel CAS9-based insertional mutation in MED21. The MED21 genomic locus (AT4G04780) is shown as a cartoon, with a zoom-in on the beginning of the coding …
Author response image 1
Tables
Key resources table
| Reagent type (species) or resource | Designation | Source or reference | Identifiers | Additional information |
|---|---|---|---|---|
| Gene (Arabidopsis thaliana) | TOPLESS, TPL | GenBank | AT1G15750 | |
| Gene (Arabidopsis thaliana) | MEDIATOR 21, MED21 | GenBank | AT4G04780 | |
| Strain, strain background (Saccharomyces cerevisiae) | Anchor Away strains | EURO- SCARF euroscarf.de | HHY168 | See Yeast Strain list (Supplementary file 3) |
| Strain, strain background (Saccharomyces cerevisiae) | cytoSUS strains | Asseck and Grefen, 2018 | THY.AP4, THY.AP5 | See Yeast Strain list (Supplementary file 3) |
| Strain, strain background (Escherichia coli) | Rosetta 2 strain | Sigma-Aldrich | 71400 | Electrocompetent cells |
| Strain, strain background (Nicotiana benthamiana) | Nicotiana benthamiana (wild-type) | GenBank | NCBI: txid4100 | |
| Strain, strain background (Agrobacterium tumefaciens) | GV3101 | GenBank | NCBI: txid358 | Electrocompetent cells |
| Genetic reagent (Arabidopsis thaliana) | J0121 (in Col-0 accession) | Gala et al., 2021 | J0121 | |
| Genetic reagent (Arabidopsis thaliana) | slr | TAIR | SLR-1, AT4G14550 | |
| Genetic reagent (Arabidopsis thaliana) | med21-1 | Arabidopsis Biological Resource Center | WiscDsLox461-464K13 | |
| Genetic reagent (Arabidopsis thaliana) | med21i214G | This paper | ||
| Antibody | Anti-HA-HRP (Rat Monoclonal) | Roche/Millipore Sigma | RRID:AB_390917, REF-12013819001, Clone 3F10 | WB (1:1000) |
| Antibody | Anti-FLAG (Mouse Monoclonal) | Millipore Sigma | RRID:AB_259529, F3165 | WB (1:5000) |
| Antibody | Anti-FRB (Rabbit Polyclonal) | Enzo Life Sciences, (Haruki et al., 2008) | RRID:AB_2051920, ALX-215-065-1 | WB (1:10,000) |
| Antibody | Anti-VP16 (1-21) (Mouse Monoclonal) | Santa Cruz Biotechnology | RRID:AB_628443, sc-7545 | WB (1:5000) |
| Antibody | Anti-GFP (Rabbit Polyclonal) | AbCam | RRID:AB_303395, ab290 | WB (1:10,000) |
| Antibody | Anti-MYC (Rabbit Monoclonal) | Cell Signaling | RRID:AB_490778, 71d10, 2278S | WB (1:5000) |
| Antibody | Anti-PGK1 (Mouse Monoclonal) | AbCam | RRID:AB_10861977, ab113687 | WB (1:10,000) |
| Recombinant DNA reagent | RPL13A-FKBP fusion proteins | Haruki et al., 2008 | See Plasmid list (Supplementary file 2) | |
| Peptide, recombinant protein | TPL-6xH | This paper | TPL-6xHis tagged fusion proteins | |
| Commercial assay or kit | DNA Sequencing | Genewiz | Genewiz.com | |
| Chemical compound, drug | Rapamycin | LC Laboratories | R-5000 | 1 µM for Anchor Away |
| Chemical compound, drug | β-estradiol | Sigma | E2758-1G | |
| Chemical compound, drug | Auxin | plantMedia, plantmedia.com | CAT#705490 | (IAA-10 µM) |
| Chemical compound, drug | Geneticin | Thermo Fisher Scientific | G418 | |
| Software, algorithm | CLC Sequence Viewer 7 | QIAGEN | ||
| Software, algorithm | R | R Studio | rstudio.com/ | |
| Software, algorithm | ImageJ | Schneider et al., 2012 | https://imagej.nih.gov/ij/ | |
| Software, algorithm | SmartRoot | Jülich Research Centre and ROot and Soil/Shoot Interactions virtual group | https://smartroot.github.io/ | |
| Software, algorithm | NeuronJ | Erik Meijering | https://imagescience.org/meijering/software/neuronj/manual/ |
Additional files
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Source code 1
Custom scripts.
Custom scripts used in this study in an R file format to analyze flow cytometry and to quantify root phenotypes. Comments are included to delineate sections in the code. Also available on Github: https://github.com/achillobator/TPL_Structure_Function/.
- https://cdn.elifesciences.org/articles/66739/elife-66739-code1-v2.zip
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Supplementary file 1
Oligonucleotide list.
Sequences, names, and experimental uses of all oligonucleotides created in this study.
- https://cdn.elifesciences.org/articles/66739/elife-66739-supp1-v2.xlsx
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Supplementary file 2
Plasmid list.
Names and descriptions of all plasmids generated in this study.
- https://cdn.elifesciences.org/articles/66739/elife-66739-supp2-v2.xlsx
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Supplementary file 3
Yeast strain list.
Names and full genotypes of all yeast strains generated or used in this study.
- https://cdn.elifesciences.org/articles/66739/elife-66739-supp3-v2.xlsx
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Transparent reporting form
- https://cdn.elifesciences.org/articles/66739/elife-66739-transrepform-v2.docx
