The program of spatial and temporal gene expression initiated upon reception of the Hedgehog (Hh) signal is regulated by the transcription factor Cubitus interruptus (Ci). Misregulation of Ci activity can result in developmental defects and cancer, and the Hh signaling pathway has evolved multiple layers of posttranslational regulation of Ci (such as repressor formation, protein degradation, and subcellular localization) to prevent both underactivation and hyperactivation of Hh target gene transcription and ensure proper patterning. The work described identifies and analyzes three distinct regulatory domains of Ci, using mutational analysis of transgenic Ci constructs and characterization with an in vivo wing disc assay and immunoblotting in the fruit fly, Drosophila melanogaster.
One domain, the RFD (Repressor Formation Domain), is required but not sufficient for Ci repressor (Ci-75) formation. The RFD is distinct from the site of cleavage and has homology to the vertebrate Gli proteins that are cleaved, Gli2 and Gli3. My results also support an alternative model for Ci-75 formation in which Supernumerary limb targets Smoothened for degradation, allowing cleavage of Ci by an exo-site protease.
A second domain, comprising residues 1160-1351, is downstream of the only identified activation domain in Ci, the CBP interaction domain. Using both tissue culture and in vivo expression in Drosophila I show that this region is involved in destabilizing Ci protein, regulating Ci protein turnover, and thereby preventing its transformation into Ci activator (Ci-act).
The third domain, consisting of residues 1352-1398, has significant homology to Gli2 and Gli3, and is called the Gli Homology Domain (GHD). Using clonal analysis in the Drosophila wing disc and staining in salivary glands, I demonstrate that the GHD is crucial for both Ci activation and nuclear export.