An EcR probe reveals mechanisms of the ecdysone-mediated switch from repression-to-activation on target genes in the larval wing disc

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 (EcRLBD) as a sponge to sequester coregulators and probe the state of EcR activity as larval wing cells traverse the 3rd 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 Ala483Thr mutation that disrupts binding of the co-repressor Smr compromises the ability of EcRLBD 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 EcRLBD sequesters 20E, and that EcRLBD 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 EcRLBD as an effective probe to define EcR-20E gene regulatory mechanisms in vivo.