Projet ANR-07-BLAN-0071 – FUNCTIONAL : : Functional characterisation of new genes involved in the secretion and release of cholesterol-modified Hedgehog.
Programme : Blanc (BLANC) 2007
Année de financement : 2007
Coordinateur du projet : Monsieur PASCAL THEROND (CNRS DELEGATION REGIONALE COTE D'AZUR)
Partenaires : Centre national de la recherche scientifique
Montant : 300000.0 euros
Date de début : 2007
Durée : 36 mois
Résumé : Hedgehog (Hh) familly protein are secreted molecules that function as organisers in animal development (Ingham and McMahon, 2001). Moreover, Hh signaling appears to regulate stem cell homeostasis in adult tissues, while persistent Hh pathway activity has pathological consequences in a number of cancers (Beachy et al., 2004). Hh familly proteins are the only known metazoan proteins with a covalently-linked cholesterol moiety. Therefore, the cellular mechanisms that has evolved to handle lipidated Hh proteins to be deployed in normal developing and tumourigenic tissues have become under intense scrutiny. Our objective is to understand how cholesterol-modified Hh is secreted and released from the producing cells. In view of the unique features of Hh, we hypothesize that a very specific cellular activity is necessary for Hh secretion and release. Our goal is to screen on a genome-wide scale for genes involved in Hh secretion with the use of the RNA interference (RNAi)-mediated disruption of gene function. A library of double-stranded RNA (dsRNA) directed against the all predicted open reading frames in Drosophila will be used to conduct robotized high-throughput cell-based RNAi screens. This library of dsRNA will be screened for presence of secreted Hh in cell cultured conditioned medium. To quantify directly the release of processed Hh proteins, the coding sequence of the Renilla luciferase was fused in frame with Hh full length sequence. Pilot experiments showed that secretion of active Hh-renilla fusion can be modulated by dsRNA treatments. Once the screen completed the candidate genes will be compared with genes identified in two other RNAi screens: one on the general secretion machinery and another on the regulation of the secretion of Wingless, another lipidated protein. The comparison of these different screens will allow us to identify specific functions involved in cholesterol-modified Hh secretion and release. In vivo validation will be developped with the use of a unique collection of inducibleRNAi transgenic lines allowing tissue-targeted gene silencing. By identifying and analysing these new genes we will gain a greater understanding of how Hh is secreted. Such genes may represent the starting point to develop new diagnostic tools for Hh-dependent pathologies and new ways of correcting Hh signal in Hh-dependent tumours driven by endogenous expression of Hh ligands.                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                        ...