Repression by the Arabidopsis TOPLESS corepressor requires association with the core Mediator complex
- University of Washington, United States
Abstract
The plant corepressor TOPLESS (TPL) is recruited to a large number of loci that are selectively induced in response to developmental or environmental cues, yet the mechanisms by which it inhibits expression in the absence of these stimuli is poorly understood. Previously, we had used the N-terminus of Arabidopsis thaliana TPL to enable repression of a synthetic auxin response circuit in Saccharomyces cerevisiae (yeast). Here, we leveraged the yeast system to interrogate the relationship between TPL structure and function, specifically scanning for repression domains. We identified a potent repression domain in Helix 8 located within the CRA domain, which directly interacted with the Mediator middle module subunits Med21 and Med10. Interactions between TPL and Mediator were required to fully repress transcription in both yeast and plants. In contrast, we found that multimer formation, a conserved feature of many corepressors, had minimal influence on the repression strength of TPL.
Data availability
All flow cytometry data is deposited at https://flowrepository.org/. Repository IDS: FR-FCM-Z2GM, FR-FCM-Z2GR, FR-FCM-Z2GQ, FR-FCM-Z2GX, FR-FCM-Z2GT, FR-FCM-Z2W2. All protein interactions have been deposited to IntAct, under the accession code, IM-28972. All code is available through Github: https://github.com/achillobator/TPL_Structure_Function/
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TPL Helix-By-Helix deletion seriesFlow Repository, FR-FCM-Z2GM.
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TPL Helix-By-Helix deletion series 2Flow Repository, FR-FCM-Z2GR.
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TPL Helix-By-Helix deletion series 3Flow Repository, FR-FCM-Z2GT.
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TPL Helix-By-Helix deletion series 4Flow Repository, FR-FCM-Z2W2.
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Repression by the Arabidopsis TOPLESS corepressor requires association with the core Mediator complexEBI IntAct Molecular Interaction Database, IM-28972.
Article and author information
Author details
Alexander R Leydon
Jennifer L Nemhauser
Funding
National Institutes of Health (GM107084)
- Alexander R Leydon
- Hardik P Gala
- Sabrina Gilmour
- Samuel Juarez-Solis
- Mollye L Zahler
- Joseph E Zemke
- Jennifer L Nemhauser
Howard Hughes Medical Institute
- Alexander R Leydon
- Wei Wang
- Hardik P Gala
- Sabrina Gilmour
- Samuel Juarez-Solis
- Mollye L Zahler
- Joseph E Zemke
- NIng Zheng
- Jennifer L Nemhauser
The funders had no role in study design, data collection and interpretation, or the decision to submit the work for publication.
Copyright
© 2021, Leydon et al.
This article is distributed under the terms of the Creative Commons Attribution License permitting unrestricted use and redistribution provided that the original author and source are credited.
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Repression by the Arabidopsis TOPLESS corepressor requires association with the core Mediator complex
eLife 10:e66739.
https://doi.org/10.7554/eLife.66739
Further reading
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Type II nuclear receptors (T2NRs) require heterodimerization with a common partner, the retinoid X receptor (RXR), to bind cognate DNA recognition sites in chromatin. Based on previous biochemical and overexpression studies, binding of T2NRs to chromatin is proposed to be regulated by competition for a limiting pool of the core RXR subunit. However, this mechanism has not yet been tested for endogenous proteins in live cells. Using single-molecule tracking (SMT) and proximity-assisted photoactivation (PAPA), we monitored interactions between endogenously tagged RXR and retinoic acid receptor (RAR) in live cells. Unexpectedly, we find that higher expression of RAR, but not RXR, increases heterodimerization and chromatin binding in U2OS cells. This surprising finding indicates the limiting factor is not RXR but likely its cadre of obligate dimer binding partners. SMT and PAPA thus provide a direct way to probe which components are functionally limiting within a complex TF interaction network providing new insights into mechanisms of gene regulation in vivo with implications for drug development targeting nuclear receptors.
