Role of Capicua-L in RTK signaling and endocycle regulation

Abstract

The HMG-box protein Capicua (Cic) is a conserved transcriptional repressor with key functions downstream of receptor tyrosine kinase (RTK)-Ras-MAPK signaling pathways. In both Drosophila and mammals, Cic is expressed as short (Cic-S) and long (Cic-L) isoforms that differ in their N-terminal regions. However, the significance of this difference or whether Cic-S and Cic-L have different functions or regulation is not known. This is because most of the work carried out so far has been done on Cic-S or using approaches that do not discriminate between both isoforms. To address this question, we have compared the expression of both isoforms during Drosophila development and obtained CRISPR-induced mutations specifically affecting Cic-L. We find that Cic-L acts redundantly with Cic-S in RTK processes such as wing vein specification and dorsoventral patterning of the embryo. In addition, Cic-L exerts individual functions regulating germline cell growth and development during oogenesis. Specifically, Cic-L accumulates in nurse cell nuclei during mid-oogenesis and is necessary for nurse cell endocycle termination and massive transfer or “dumping” of nurse cells contents into the oocyte in late oogenesis. In addition, we show that endocycle exit precedes the initiation of nurse cell dumping and propose that Cic-L enables nurse cell dumping by triggering endocycle exit. Cic-L exerts this control, at least in part, by promoting stabilization of Cyclin E, a key regulator whose periodic oscillations drive endoreplicative cycles, and downregulation of the Myc cell-growth factor. We also find that these unique functions of Cic-L primarily depend on its specific N-terminal module, which contains three conserved domains –NLS, Tudor-like and N1– that contribute additively to function. In contrast, other domains shared with Cic-S –the HMG- box and C1 DNA binding domains and the C2 motif necessary for MAPK-dependent downregulation– are largely dispensable for Cic-L-specific activity. Finally, we note that basal metazoans including sponges possess truncated “Proto-Cic” variants composed only of Cic-L N-terminal sequences, without the characteristic Cic HMG-box domain. Thus, the Cic-L N-terminal region plays unexpected roles in cell growth and endoreplication that may resemble the ancestral activities of Cic-like proteins in evolution.