Regulation of Drosophila FKH/FOXA by TOR Figure 6. FKH is required for the response to rapamycin and lowered TOR levels. Realtime qPCR was performed to quantify mRNAs in larval extracts. (A) Transcription of CG6770 and cabut is induced upon rapamycin treatment (Rapa) and downregulated in larvae with low FKH levels (w; arm-Gal4/+; UAS-FKH-RNAi/+). Compared to rapamycin-fed wildtype and unspecific control animals (w; arm-Gal4/+; UAS-LacZ-RNAi/+), expression of CG6770 and cabut is significantly lower in rapamycin-fed FKH-RNAi larvae. (B) Consistent with the high expression of CG6770 and cabut in rapamycin-treated larvae, these genes are transcriptionally upregulated in dTOR mutants (y w; dTORDP/+ and y w; dTORDP/dTORDP). The elevated expression is suppressed in larvae transheterozygous for dTOR and FKH (y w; dTORDP/+; fkh1/+ and y w; dTORDP/+; fkh6/+), indicating that FKH function is required for target gene induction by low TOR signaling. (C) The body weight of adult flies heterozygous for dTORDP is increased by the presence of one copy of the fkh1 allele. Both male and female transheterozygous flies are slightly but significantly heavier than dTORDP/+ flies. 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.g006 alleles, corroborating the findings obtained from our RNA inter- ference experiments. In addition to its function in the regulation of TOR-dependent growth and gene expression, we investigated the impact of nutrient levels and TOR signaling on the subcellular localization of FKH. On a protein-rich-diet, FKH was excluded from the nucleus in larval fatbody tissue, and inhibition of TOR signaling by pharmacological or genetic means elicited a robust nuclear translocation of the transcription factor. Starvation induced by complete nutrient deprivation on PBS only had a weak effect on FKH localization, diminishing nuclear exclusion to the point that the contours of the nuclei, which were visible due to the absence of nuclear signal in the yeast feeding condition, were no longer discernible. Albeit weak, we believe that this increase in nuclear localization is biologically relevant, as FKH function is required for the starvation-induced attenuation of cell growth in the larval fatbody (Figures 2 and 3). As described above, the subcellular distribution of FKH in starved animals is in stark contrast to the one of dFOXO, which is predominantly nuclear in this nutritional state. Another apparent difference between the shuttling of FKH and dFOXO is that in the case of dFOXO, both the overexpressed and endogenous protein translocates to the cytoplasm upon insulin stimulation of cells. This likewise holds true for the mammalian FOXO proteins. The situation for FOXA proteins appears to be somewhat less uniform. We report for the fly FOXA/FKH that the endogenous protein is excluded from the nuclei in the larval fatbody under conditions of highly abundant dietary protein and amino acids, which translates into high TOR signaling activity. In contrast, overexpressed FKH protein is constitutively nuclear even on a high protein diet. The cause for this may be that the abundance of one or several upstream regulators is limiting and tuned to endogenous FKH concentrations. If the copy number of FKH protein is elevated beyond physiological levels, it may escape negative regulation and nuclear export induced by the endogenous signaling components, and thus accumulate in the PLoS ONE | www.plosone.org 8 December 2010 | Volume 5 | Issue 12 | e15171