Regulation of Drosophila FKH/FOXA by TOR Figure 5. Transcription of CG6770 and cabut is regulated by FKH and rapamycin. Realtime qPCR was performed to quantify mRNAs in larval extracts. (A) Compared to wildtype animals, the putative FKH targets CG6770 and cabut are downregulated in yeast-fed larvae with low FKH levels (w; arm-Gal4/+; UAS-FKH-RNAi/+) and upregulated in larvae overexpressing FKH (w;; ppl-Gal4/UAS-FKH). (B) CG6770 and cabut transcription is induced in wildtype larvae fed with 50 mM rapamycin. (C) CG6770 and cabut mRNA levels are significantly elevated upon rapamycin treatment and in larvae with high FKH levels in comparison to the wildtype and unspecific controls (w;; ppl-Gal4/UAS-LacZ). Rapamycin treatment of larvae with high FKH levels has no significant additive effect on target gene expression. Significance was tested using an unpaired 2-tailed Student’s t-test. * = p,0.05; ** = p,0.01; *** = p,0.001. Error bars represent SEM. doi:10.1371/journal.pone.0015171.g005 of d4E-BP, which in turn represses cap-dependent translation under conditions of nutrient scarcity [30,31]. Rapamycin was found to induce transcription of d4E-BP in larvae, whereas RNAi-knock- down of FKH lead to a significant decrease of expression under both conditions. Conversely, overexpression of FKH elicited a strong elevation of d4E-BP mRNA levels (Figure 7A). Similar to the effect on CG6770 and cabut, TOR-mutant larvae displayed increased d4E- BP transcription, which was completely suppressed by the fkh1 or the fkh6 mutation (Figure 7B). These results suggest that induction of d4E-BP transcription is not only mediated by dFOXO under conditions of starvation and low insulin signaling, but also by FKH when TOR signaling levels are low. Discussion In C. elegans, the FoxA transcription factor PHA-4 has been identified as a mediator of TOR signaling in the regulation of lifespan. In this study, we present a characterization of the fly FoxA protein FKH in the biological context of TOR-regulated growth and gene transcription. Figure 8 shows a simplified schematic representation of the working model which we propose based on our experimental results, describing FKH as a mediator of the cellular response to protein deprivation. As FKH has been previously uncharacterized in terms of growth control, we first established that alterations of FKH levels lead to changes in cellular and organismal size. Ectopic expression of FKH reduced growth of feeding larvae and of fatbody cells under conditions of nutrient abundance, and induced expression of the rapamycin-sensitive genes CG6770 and cabut. The observation that FKH overexpression phenotypes are strongest under conditions of dietary protein abundance and active TOR signaling might reflect a scenario in which the abundance of upstream negative regulators of FKH is limiting under these conditions. Thus an experimental elevation of FKH protein levels would override the endogenous upstream signaling systems and elicit an inhibition of growth. As discussed below, the same argumentation could explain the finding that in contrast to the endogenous protein, overexpressed FKH accumulates in the nucleus even on a high-protein diet. The same three readouts were used to demonstrate that FKH function is required for growth inhibition downstream of TORC1. Targeted knockdown of FKH by RNAi diminished the decrease in larval size, fatbody cell size as well as the induction of rapamycin target gene expression when TOR signaling activity was lowered. The elevated transcription of CG6770, cabut and d4E-BP in heterozy- gous TOR mutants was likewise suppressed by fkh loss-of-function PLoS ONE | www.plosone.org 7 December 2010 | Volume 5 | Issue 12 | e15171