by low FKH levels and elevated upon FKH overexpression. Moreover, 4E-BP transcript levels are elevated upon pharmaco- logical or genetic inactivation of TOR. As with the FKH target genes cabut and CG6770, the positive effect of rapamycin or TOR mutation on 4E-BP expression is suppressed by FKH knockdown or FKH loss-of-function mutations. We therefore argue that d4E- BP is most likely a common transcriptional target of both dFOXO and FKH and thus constitutes an additional point of crosstalk between the insulin and TOR signaling modules. The fact that FKH and dFOXO share the conserved forkhead DNA binding domain may suggest at least partially overlapping target gene populations. Regarding the growth-suppressive action of FKH under conditions of nutrient deprivation, it is currently unclear whether growth inhibition is mainly achieved through the induction of CG6770, cabut and d4E-BP or if other, yet unidentified FKH targets also contribute to this phenotype. d4E-BP mutants have no overgrowth phenotype under normal conditions [37], but it remains to be investigated whether they display impaired growth inhibition under starvation conditions. The observation that the Thor1 allele partially suppresses the Akt1 cell number phenotype in the eye provides evidence that d4E-BP function is required when insulin signaling levels are lowered [30]. In cultured cells, knockdown of CG6770 [29] or cabut [25] leads to increased cell size, raising the possibility that they also act as negative regulators of growth downstream of FKH. Further studies are required to assess the relative relevance of the individual target genes in this context. In summary, we present the first evidence that the interaction between TOR signaling and FoxA proteins is conserved in Drosophila. Our study supports a model in which FKH is regulated by the TOR pathway and dFOXO by the insulin/PI3K pathway, two signaling systems that are already interwoven at several points such as TSC2 and 4E-BP. On the level of transcription factors, there seems to be differential regulation: FKH is activated under conditions of protein deprivation and low TOR signaling, while dFOXO is activated by complete starvation and low insulin signaling. However, there is also a downstream node of conver- gence. The expression of the translational inhibitor d4E-BP, which has been established as a dFOXO target and is transcriptionally induced by protein deprivation as well as complete starvation [27], is induced under conditions of low TOR signaling by FKH. This emerging molecular scenario outlined by these observations would allow cells and organisms to react specifically to different conditions of nutrient availability and food composition. Materials and Methods Constructs, antibodies and fly lines The open reading frames (ORFs) encoding FKH (a protein comprising 510 amino acid residues) and dFOXO (a protein comprising 613 amino acid residues) were PCR-amplified from genomic DNA (fkh is a single-exon gene) isolated from adult flies and from a pUAST-dFOXO plasmid template [30], respectively. The amplicons were cloned into pENTR/D-TOPO (Invitrogen) to generate gateway entry constructs, which were fully sequenced to ensure sequence integrity. Cell culture expression constructs were then created by transferring the ORFs via LR recombination into the modified gateway destination vector pAHW-Blast, which corresponds to the pAHW plasmid from Terence Murphy’s Drosophila Gateway Vector Collection [38] with a blasticidin resistance cassette cloned into the backbone, and allows expression of N-terminally 3xHA-tagged proteins under control of the Actin5C promoter. Furthermore, a pAGW-FKH construct was generated to allow expression of a green fluorescent EGFP-FKH fusion protein. P-element based expression constructs for trans- Regulation of Drosophila FKH/FOXA by TOR genic flies were created by transferring the fkh ORF into pTHW and pTRW from the same vector collection. The resulting plasmids pTHW-FKH and pTRW-FKH allow GAL4-induced expression of 3xHA-FKH and red fluorescent mRFP1-FKH, respectively. An inverted repeat construct for the targeted in vivo knockdown of FKH by RNAi was created by first PCR-amplifying the first 701 bp of the fkh ORF with a XhoI linker added to the forward and a BglII linker to the reverse primer. The resulting amplicon was digested with XhoI and then self-ligated with T4 DNA ligase. The correct 1.4 kb ligation product was gel purified, digested with BglII and cloned into the BglII site of pMF3 [39]. The cloning step was performed in SURE bacteria (Stratagene). The CG6770 reporter plasmid pGL3-CG6770 was constructed by cloning a 880 bp PCR amplicon covering the CG6770 regulatory region up to the translation start codon into the pGL3-Basic vector containing the gene encoding Photinus pyralis (firefly) luciferase (Promega) as a BglII/blunt fragment. pGL3-Basic was first digested with NcoI, the resulting 59 overhangs were then blunted with T4 DNA polymerase before proceeding with BglII digestion as the second step. The PCR was performed with a 59- phosphorylated reverse primer to ensure efficient ligation by T4 DNA ligase. The control construct for expressing Renilla reniformis luciferase under control of the RpIII128 promoter, polIII-RL [40], was a kind gift of Norbert Perrimon. Phusion DNA polymerase (Finnzymes) was used for all PCR steps in this study. All primer sequences are provided as supporting information in Text S1. A polyclonal antibody against FKH was generated by Coring Systems Diagnostix (Gernsheim, D). aFKH1 was raised in rabbit against the peptide SHSSLEATSPGKKD, and purified by immunoaffinity chromatography. The location of the peptide in the FKH protein sequence is provided as supporting information in Text S3. Two independent transgenic UAS-FKH-RNAi lines were mainly used. One was established with our pMF3-FKH construct described above, and one was obtained from the Transgenic RNAi Project (TRiP) at Harvard Medical School (TRiP stock JF02417) [41]. A third construct which was generated by cloning a 913 bp amplicon spanning part of the fkh ORF and part of the fkh 39-UTR into the Sym-pUAST-w vector [42] was found to be less effective in inducing in vivo knockdown compared to the other two constructs (data not shown), and was therefore not further used in most experiments. The regions in the fkh mRNA which are targeted by the three individual RNAi constructs are provided as supporting information in Text S2. All in vivo RNAi experiments which involved knockdown of FKH expression were performed in parallel with transgenics carrying the pMF3 construct or the TRiP construct, and identical phenotypes were observed for both. The loss-of-function allele dTORDP, which is a deletion generated by imprecise P-element excision and removes the dTOR translation start site as well as the amino-terminal 902 codons [14], was a gift of Tom Neufeld. The fkh loss-of-function alleles fkh1 (stock nr. 3331) and fkh6 (stock nr. 545) were obtained from the Bloomington Drosophila Stock Center. fkh1 contains an ethyl methanesulfonate (EMS)-induced point mutation changing the codon for W254, a residue in the forkhead DNA-binding domain, into a stop codon, while the X-ray-induced allele fkh6 carries an 11 bp deletion which produces a frameshift after the codon for residue E7 [15]. FKH overexpression in vivo was achieved by use of transgenic UAS-3xHA-FKH or UAS-mRFP1- FKH lines which were established with our pTHW-FKH and pTRW-FKH constructs. Larval starvation and rapamycin feeding If not stated otherwise, embryos were collected for 4 h on PBS- agar plates with yeast paste (prepared by suspending one cube of PLoS ONE | www.plosone.org 10 December 2010 | Volume 5 | Issue 12 | e15171