An EcR probe reveals mechanisms of the ecdysone-mediated switch from repression-to-activation on target genes in the larval wing disc
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The manuscript by Wardwell-Ozgo and co-authors describes a thorough and interesting study that explores the mechanisms through which a hormone receptor can both repress and activate gene transcription. They have conducted an impressive number of experiments all aimed at showing that by using their new transgenic tool, and Ecdysone Receptor (EcR) ligand binding domain sponge, they can demonstrate that EcR activity is important for eliciting both types of ecdysone responses, repression, and activation, in the Drosophila wing disc and that the EcR binding partner Smarter is essential for the repressive function. The differences in expression levels have however not been quantified, which would lend greater support to their claims.
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Abstract
Fluctuating levels of steroid hormones provide both systemic and local cues to synchronize metazoan development and control germline and homeostatic processes. The main steroid hormone in Drosophila is ecdysone (Ec), which upon binding of its active form (20E) converts its receptor, EcR, from a transcriptional repressor to activator. Multiple co-repressors and co-activators are proposed to act with EcR in different tissues to control diverse targets and processes, including apoptosis, cell migration, and proliferation. Despite these diverse roles, relatively little is known regarding how EcR translates Ec temporal gradients into modulation of individual target genes. Here we use an Ec-binding fragment of EcR (EcR LBD ) as a ‘sponge’ to sequester coregulators and probe the state of EcR activity as larval wing cells traverse the 3 rd instar Ec gradient. This approach reveals a dramatic and rapid shift from EcR mediated repression-to-activation in late L3 cells, and that the extent of repression varies between targets. An Ala 483 Thr mutation that disrupts binding of the co-repressor Smr compromises the ability of EcR LBD to derepress reporters, but also limits its ability to block activation, suggesting either that a coactivator shares an EcR-interaction interface with Smr or that Smr-repression primes targets for 20E activation. Molecular and genetic data reveal that EcR LBD sequesters 20E, and that EcR LBD phenotypes can be modulated by manipulating intracellular 20E levels with Ec importer (EcI) and Cyp18a1, which inactivates 20E. Finally, we provide evidence that Smr repression of EcR activity varies spatially and by target in the wing disc. In sum these data reveal that relief of EcR-Smr repression is a major contributor to 20E induction of EcR targets in larval wing discs and highlight EcR LBD as an effective probe to define EcR-20E gene regulatory mechanisms in vivo .
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eLife assessment
The manuscript by Wardwell-Ozgo and co-authors describes a thorough and interesting study that explores the mechanisms through which a hormone receptor can both repress and activate gene transcription. They have conducted an impressive number of experiments all aimed at showing that by using their new transgenic tool, and Ecdysone Receptor (EcR) ligand binding domain sponge, they can demonstrate that EcR activity is important for eliciting both types of ecdysone responses, repression, and activation, in the Drosophila wing disc and that the EcR binding partner Smarter is essential for the repressive function. The differences in expression levels have however not been quantified, which would lend greater support to their claims.
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Reviewer #1 (Public Review):
In this manuscript, the authors explore the mechanisms through which hormone receptors act on their targets to either repress or activate transcription. To do this, they employ a new transgenic tool, a transgenic construct that contains only the ligand binding domain for the ecdysone receptor, EcRLBD, that acts as a sponge for both the steroid hormone ecdysone and for EcR-binding partners. They find that their EcRLBD elicits many of the same phenotypes as other tools used to manipulate the EcR function, suggesting that it acts as a dominant negative. However, it does not elicit all of the same phenotypes as EcR RNAi or overexpression of other dominant negative EcR transgenes (EcRF645A). For example, it interferes with fat body mobilization into the pupal head but does not affect the disintegration of the …
Reviewer #1 (Public Review):
In this manuscript, the authors explore the mechanisms through which hormone receptors act on their targets to either repress or activate transcription. To do this, they employ a new transgenic tool, a transgenic construct that contains only the ligand binding domain for the ecdysone receptor, EcRLBD, that acts as a sponge for both the steroid hormone ecdysone and for EcR-binding partners. They find that their EcRLBD elicits many of the same phenotypes as other tools used to manipulate the EcR function, suggesting that it acts as a dominant negative. However, it does not elicit all of the same phenotypes as EcR RNAi or overexpression of other dominant negative EcR transgenes (EcRF645A). For example, it interferes with fat body mobilization into the pupal head but does not affect the disintegration of the larval fat body sheets as do the EcR RNAi or EcRF645A.
The authors proceed to provide extensive evidence that the EcRLBD affects both the repression and activation functions of EcR, using EcRE lacz and EcRE GFP transgenes in the developing wing disc. Modifying 20E uptake or metabolism does not affect the ability of EcRLBD to induce precocious de-repression. This is perhaps unsurprising as EcRLBD is proposed to be sponging co-repressors which would be necessary for unliganded EcR repression. However, reducing 20E metabolism does rescue some of the effects of EcRLBD on the activation of gene expression.
The EcRLBD can also induce precocious de-repression of key ecdysone response genes Broad and E93. However, neither of these genes appear to require EcR activation as the later-stage expression is not reduced in EcRLBD larvae. Finally, they demonstrate that the effects they observe when overexpressing EcRLBD in a variety of tissues depend on the ability to bind to a co-repressor Smarter.
There is an impressive amount of work in this manuscript, and the data appears to be of high quality. The experiments are appropriate to the authors' aims, and I feel they will be of broad interest to all those working on developmental physiology and receptor/hormone interactions. Their new transgenic tool is sure to be used by a number of researchers interested in identifying binding partners for EcR across developmental timescales.
I think the most significant weakness of this work is none of the data has been quantified and so it's difficult to judge the extent of variation in samples. Quantification is important, as many of the arguments are based on relative levels of expression. While I feel that the study design supports the authors' aims, the lack of quantitative analysis limits the extent to which the data supports their conclusions.
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Reviewer #2 (Public Review):
In this work, the authors analyze the mechanism through which the fluctuations of the Ecdysone hormone modulate the passage from a third instar larva to a pupa, during the process of metamorphosis. They focus on the imaginal wing disc in which initially the levels of Ecdysone fluctuate and in the later phase when the levels of this hormone increase significantly. This entire process depends on the Ecdysone hormone receptor (EcR) and the interaction it has with co-repressors and co-activators. Using as a tool a dominant negative form that does not have the receptor DNA binding site, but does have the hormone binding site as well as regions with which the receptor interacts with co-repressors and co-activators, they show that genes which are repressed early in the wing disc, are de-repressed if this dominant …
Reviewer #2 (Public Review):
In this work, the authors analyze the mechanism through which the fluctuations of the Ecdysone hormone modulate the passage from a third instar larva to a pupa, during the process of metamorphosis. They focus on the imaginal wing disc in which initially the levels of Ecdysone fluctuate and in the later phase when the levels of this hormone increase significantly. This entire process depends on the Ecdysone hormone receptor (EcR) and the interaction it has with co-repressors and co-activators. Using as a tool a dominant negative form that does not have the receptor DNA binding site, but does have the hormone binding site as well as regions with which the receptor interacts with co-repressors and co-activators, they show that genes which are repressed early in the wing disc, are de-repressed if this dominant negative is present. Even more, they manage to demonstrate that at the genetic level, one of the co-repressors that acts together with the EcR in the repression of these genes is Smrter/NCoR1. The strategy used is based on the use of genetic tools that are unique to Drosophila, which allows them to carry out a very precise analysis of the expression of the reporter and endogenous genes in a very fine way and allows them to obtain very robust in vivo results. On the other hand, the work can be reinforced using biochemical techniques that may allow showing the direct interactions of the different players studied in this work. Nuclear receptors that respond to steroid hormones are present in all metazoa. Therefore, this work is useful not only to understand the mechanisms of how nuclear receptors modulate gene expression in flies but also in mammals.
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