The Drosophila FoxA Ortholog Fork Head Regulates Growth and Gene Expression Downstream of Target of Rapamycin Margret H. Bu¨ low1,2,3, Ruedi Aebersold1,3,4, Michael J. Pankratz2*, Martin A. Ju¨ nger1* 1 Institute of Molecular Systems Biology, Swiss Federal Institute of Technology (ETH) Zurich, Zurich, Switzerland, 2 Department of Molecular Brain Physiology and Behavior, Life and Medical Sciences (LIMES) Institute, University of Bonn, Bonn, Germany, 3 Competence Center for Systems Physiology and Metabolic Diseases (CC- SPMD), Swiss Federal Institute of Technology (ETH) Zurich, Zurich, Switzerland, 4 Faculty of Science, University of Zurich, Zurich, Switzerland Abstract Forkhead transcription factors of the FoxO subfamily regulate gene expression programs downstream of the insulin signaling network. It is less clear which proteins mediate transcriptional control exerted by Target of rapamycin (TOR) signaling, but recent studies in nematodes suggest a role for FoxA transcription factors downstream of TOR. In this study we present evidence that outlines a similar connection in Drosophila, in which the FoxA protein Fork head (FKH) regulates cellular and organismal size downstream of TOR. We find that ectopic expression and targeted knockdown of FKH in larval tissues elicits different size phenotypes depending on nutrient state and TOR signaling levels. FKH overexpression has a negative effect on growth under fed conditions, and this phenotype is not further exacerbated by inhibition of TOR via rapamycin feeding. Under conditions of starvation or low TOR signaling levels, knockdown of FKH attenuates the size reduction associated with these conditions. Subcellular localization of endogenous FKH protein is shifted from predominantly cytoplasmic on a high-protein diet to a pronounced nuclear accumulation in animals with reduced levels of TOR or fed with rapamycin. Two putative FKH target genes, CG6770 and cabut, are transcriptionally induced by rapamycin or FKH expression, and silenced by FKH knockdown. Induction of both target genes in heterozygous TOR mutant animals is suppressed by mutations in fkh. Furthermore, TOR signaling levels and FKH impact on transcription of the dFOXO target gene d4E-BP, implying a point of crosstalk with the insulin pathway. In summary, our observations show that an alteration of FKH levels has an effect on cellular and organismal size, and that FKH function is required for the growth inhibition and target gene induction caused by low TOR signaling levels. Citation: Bu¨ low MH, Aebersold R, Pankratz MJ, Ju¨ nger MA (2010) The Drosophila FoxA Ortholog Fork Head Regulates Growth and Gene Expression Downstream of Target of Rapamycin. PLoS ONE 5(12): e15171. doi:10.1371/journal.pone.0015171 Editor: Dana Davis, University of Minnesota, United States of America Received August 11, 2010; Accepted October 27, 2010; Published December 31, 2010 Copyright: 2010 Bu¨ low, et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Funding: This work was supported by a research grant of the Swiss Federal Institute of Technology (ETH) Zu¨ rich (TH-17 07-1), the Swiss National Science Foundation (SNF) and the Deutsche Forschungsgemeinschaft (DFG). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Competing Interests: The authors have declared that no competing interests exist. * E-mail: juenger@imsb.biol.ethz.ch (MAJ); pankratz@uni-bonn.de (MJP) Introduction Transcription factors belonging to the winged helix/forkhead box (Fox) family are implicated in a variety of biological processes ranging from embryonic development to the regulation of metabolism, growth, cell death and organismal lifespan [1]. Members of this transcription factor family share a conserved 110- residue DNA binding domain first discovered in the Drosophila Fork Head protein, which therefore and because of structural reasons is referred to as the forkhead or winged helix domain. The family is divided into subclasses labeled with the letters A to S, and this categorization is based on amino acid sequence similarity in the forkhead domain [2]. For most Fox proteins, knowledge is scarce about how they are interfaced with upstream signaling pathways. A well characterized group is the FoxO subfamily which, among input from other pathways, is regulated by the insulin signaling module in a way that is conserved between the nematode Caenorhabditis elegans and humans. This involves direct phosphorylation by insulin-induced kinases, binding to 14-3-3 proteins and nucleocytoplasmic shuttling [3]. The insulin-forkhead connection was first described in C. elegans, where mutations in the insulin receptor gene daf-2 are completely suppressed by mutations in the FoxO transcription factor gene daf-16 [4,5]. Members of the FoxA subfamily are also important players in metabolism and regulated by insulin, but whether the exact mechanism also involves nuclear exclusion is still a matter of debate and less clear than for FoxO proteins [6]. Once again, pioneering research in C. elegans started to uncover a link between a growth control pathway and a forkhead transcription factor, this time between the worm Target of rapamycin (TOR) homolog LET-363 and the FoxA protein PHA-4. The first study in this line of evidence described the longevity of worms with reduced levels of TOR signaling. In contrast to the daf-16-dependent lifespan increase of insulin receptor mutant worms, long life conferred by low levels of TOR is not affected by daf-16 mutations [7]. This suggested that insulin and TOR signaling regulate lifespan through distinct downstream transcriptional regulators. Another condition which, similar to reduced TOR signaling, can prolong life in nematodes in a DAF-16-independent fashion is dietary restriction. LET-363 is involved in the dietary restriction response induced by mutations PLoS ONE | www.plosone.org 1 December 2010 | Volume 5 | Issue 12 | e15171