Repression by the Arabidopsis TOPLESS corepressor requires association with the core Mediator complex
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Evaluation Summary:
In this study, Leydon et al. use an elegant multi-component genetic system to address the mechanisms of repression by the Arabadopsis TOPLESS (Tpl) protein. Taking advantage of the genetic tools and knowledge of the structure of the Tpl protein, the authors determine two short alpha helical regions that act as independent repression domains, with the target of one of these domains being the N-terminal region of the Med21 subunit of the mediator complex. Experiments are presented that indicate that Tpl mediated repression involves formation of a promoter complex comprising the mediator complex along with several general transcription factors, but lacking RNA polymerase II. The experimental data comes from both heterologous experimental systems in yeast and the native plant setting and involves diverse but complementary experimental approaches that converge towards a model for gene repression. This paper will be of interest to researchers investigating the mechanisms regulating gene expression, in particular how specific protein-protein interactions repress gene expression.
(This preprint has been reviewed by eLife. We include the public reviews from the reviewers here; the authors also receive private feedback with suggested changes to the manuscript. Reviewer #1 and Reviewer #3 agreed to share their names with the authors.)
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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 domain 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.
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Reviewer #3 (Public Review):
In this manuscript, authors seek to resolve conflicting models for corepressor function using the elegant synthetic auxin response system. Auxin signaling is governed by a de-repression paradigm and is ideally suited to interrogate co-repressor function - in this case, the TOPLESS (TPL) co-repressor. Several contradicting models have been put forward for the mechanism of TPL-mediated gene repression, ranging from a requirement for protein oligomerization for activity, interaction with distinct partners, and even which regions of the protein are required for repressive activity. Leydon et al use the yeast-based synthetic auxin response system to interrogate these models using a single reporter locus, allowing for straight-forward examination of TPL function.
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Reviewer #2 (Public Review):
In this manuscript, the authors studied the specific domains of the plant A. thaliana TPL corepressor using a synthetic auxin response circuit (ARC) in the yeast S. cerevisiae that allows to monitor the repression and response to auxin of the reporter expression. Two domains of TPL corepressor that independently contribute to repression in this system were identified. Moreover, one of these domains interacts with Med21 and Med10 Mediator subunits. The authors show that this interaction is required for TPL-mediated auxin-responsive repression in plants. On the contrary to some repression models, they propose that multimerization of TPL is not required for repression mechanisms. Taken together, the work provides important information on auxin-responsive repression mechanisms involving TLP corepressor and the …
Reviewer #2 (Public Review):
In this manuscript, the authors studied the specific domains of the plant A. thaliana TPL corepressor using a synthetic auxin response circuit (ARC) in the yeast S. cerevisiae that allows to monitor the repression and response to auxin of the reporter expression. Two domains of TPL corepressor that independently contribute to repression in this system were identified. Moreover, one of these domains interacts with Med21 and Med10 Mediator subunits. The authors show that this interaction is required for TPL-mediated auxin-responsive repression in plants. On the contrary to some repression models, they propose that multimerization of TPL is not required for repression mechanisms. Taken together, the work provides important information on auxin-responsive repression mechanisms involving TLP corepressor and the Mediator complex.
A lot of work was done to analyze the TPL domains and critical residues involved in repression using ARC system, TPL interaction with Mediator using yeast cytoSUS and two-hybrid assays, completed by CoIP experiments with yeast and plant extracts. Point mutations, small deletions or Anchor Away-mediated depleted strains were used to analyze their consequences on TPL-Mediator interactions and auxin-responsive repression in artificial system in yeast and directly in plants.
The mechanism of how TPL-Mediator interaction is involved in auxin-responsive repression remains to be determine. No results were provided in the manuscript on the composition of Mediator upon auxin induction and a discussion sentence that "as supported by our synthetic system, auxin-induced removal of TPL is sufficient to induce changes in the composition of the Mediator complex" is not supported by the results. In general, the transition between transcriptionally repressed and active states was not analyzed. The authors have made considerable efforts to answer the reviewers' criticism and to include a number of new experiments and approaches. However, several points and conclusions need to be further developed and specified. In particular, CoIP experiments in plant extracts lack a negative IP control to conclude on the specificity of CoIP signal. Moreover, the relevance of ChIP experiments on yeast plasmid remains questionable and appropriate control regions (chromosomal ACT1 gene body is completely inappropriate as a background for Pol II ChIP), regulatory, core promoter and transcribed regions, as well as experiments with untagged control strains should be added. The ChIP occupancy was analyzed only in transcriptionally repressed state and essentially on a plasmid and no results are provided for transition to the active state.
Many problems with inappropriate citations for Figures or Figure panels did not facilitate the reading of the manuscript.
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Reviewer #1 (Public Review):
In this study, Leydon et al., use an elegant multi-component genetic system to address the mechanisms of repression by the Arabadopsis TOPLESS (Tpl) protein. Taking advantage of the genetic tools and knowledge of the structure of the Tpl protein the authors determine two short alpha helical regions that act as independent repression domains. They provide evidence that the target of one of these domains is the N-terminal region of the Med21 subunit of the mediator complex. Chromatin immunoprecipitation experiments, anchor-away loss of function and co-immunoprecipitation assays indicate that Tpl mediated repression involves formation of a promoter complex comprising the mediator complex along with several general transcription factors, but lacking RNA polymerase II. The authors also show that Tpl-Med21 …
Reviewer #1 (Public Review):
In this study, Leydon et al., use an elegant multi-component genetic system to address the mechanisms of repression by the Arabadopsis TOPLESS (Tpl) protein. Taking advantage of the genetic tools and knowledge of the structure of the Tpl protein the authors determine two short alpha helical regions that act as independent repression domains. They provide evidence that the target of one of these domains is the N-terminal region of the Med21 subunit of the mediator complex. Chromatin immunoprecipitation experiments, anchor-away loss of function and co-immunoprecipitation assays indicate that Tpl mediated repression involves formation of a promoter complex comprising the mediator complex along with several general transcription factors, but lacking RNA polymerase II. The authors also show that Tpl-Med21 interactions are involved in Tpl mediated repression in plants.
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Evaluation Summary:
In this study, Leydon et al. use an elegant multi-component genetic system to address the mechanisms of repression by the Arabadopsis TOPLESS (Tpl) protein. Taking advantage of the genetic tools and knowledge of the structure of the Tpl protein, the authors determine two short alpha helical regions that act as independent repression domains, with the target of one of these domains being the N-terminal region of the Med21 subunit of the mediator complex. Experiments are presented that indicate that Tpl mediated repression involves formation of a promoter complex comprising the mediator complex along with several general transcription factors, but lacking RNA polymerase II. The experimental data comes from both heterologous experimental systems in yeast and the native plant setting and involves diverse but …
Evaluation Summary:
In this study, Leydon et al. use an elegant multi-component genetic system to address the mechanisms of repression by the Arabadopsis TOPLESS (Tpl) protein. Taking advantage of the genetic tools and knowledge of the structure of the Tpl protein, the authors determine two short alpha helical regions that act as independent repression domains, with the target of one of these domains being the N-terminal region of the Med21 subunit of the mediator complex. Experiments are presented that indicate that Tpl mediated repression involves formation of a promoter complex comprising the mediator complex along with several general transcription factors, but lacking RNA polymerase II. The experimental data comes from both heterologous experimental systems in yeast and the native plant setting and involves diverse but complementary experimental approaches that converge towards a model for gene repression. This paper will be of interest to researchers investigating the mechanisms regulating gene expression, in particular how specific protein-protein interactions repress gene expression.
(This preprint has been reviewed by eLife. We include the public reviews from the reviewers here; the authors also receive private feedback with suggested changes to the manuscript. Reviewer #1 and Reviewer #3 agreed to share their names with the authors.)
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