Study of COP9 Signalosome in Suppression of Drosophila Ecdysone Signalling

Steroid hormones are vital to development and homeostasis in both insets and mammals. In Drosophila, the steroid hormone ecdysone controls major developmental transitions such as molting and metamorphosis, and regulates body size and female reproduction. Precise regulation of the induction as well as the termination of ecdysone signaling is essential for the proper ecdysone responses. Studies in our lab recently discovered that COP9 signolosome (CSN) is specifically required to inhibit ecdysone-mediated gene expression in Drosophila midpupal wing discs. CSN is a highly conserved, multi-subunit protease that regulates the activity of the Cullin-Ring ligases (CRLs) through deneddylation. In CSN mutants, midpupal disc cells fail to shut down expression of the key ecdysone-responsive gene br-Z1, resulting in ectopic specification of the sensory organ precursors and the transformation of non-innervated bristles into sensory organs at the posterior wing margin. Further analysis suggests that CSN suppresses ecdysone signaling through a novel deneddylation–dependent mechanism that impinges on the ecdysone-mediated transcriptional de-repression. In this grant, we propose first to study this novel CSN-dependent regulatory mechanism. We will test whether CSN regulates the activity or abundance of the repressive nuclear receptors and the E23 ABC transporter, and ask whether CSN is required to stabilize the DNA-binding activity of USP which is the heterodimeric partner of the ecdysone receptor EcR. In addition, we will perform RNAi screen to look for epigenetic modifiers that specifically participate in this CSN-dependent midpupal suppression. Previous study has found that CSN2, a CSN subunit, associates with thyroid receptor (TR) and functions as a transcriptional co-repressor. In the second part of this proposal, we will determine whether Drosophila CSN2 (dCSN2) associates with EcR and whether it acts as an adaptor to recruit CSN to EcR. We will also examine if CSN2, in addition to being a component in CSN, has a CSN-independent role in ecdysone signaling by analyzing the CSN2 mutant phenotypes in other ecdysone-mediated processes. CSN deneddylation activity regulates Cul3 stability in Drosophila cells and we also found that Cul3 is involved in Br-Z1 suppression in midpupal discs. Since Cul3 utilizes BTB domain proteins as the substrate acceptor, we propose to look for potential BTB domain protein in midpupal suppression by RNAi-based screen. Since both CSN and steroid hormone signaling are highly conserved through evolution, we believe the studies proposed in the grant will not only reveal novel biological functions of CSN in development, but also provide new insights into the molecular mechanisms of CSN and steroid hormone signaling in the higher organisms.

Project ID:PA10301-0663
External Project ID:NSC101-2311-B182-003-MY3