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.
Highlights
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]
For the first time in Drosophila, we describe an interaction between Target of rapamycin (TOR) and a FoxA protein, which is in agreement with the observations made in C. elegans
In a step following the demonstration that both genes are induced by Fork head (FKH), we addressed whether cabut and CG6770 transcription was responsive to TOR signaling levels, and could be used as a readout to confirm the interaction between the TOR module and FKH
Summary
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]. PHA-4 was recently identified as the forkhead transcription factor which is necessary to increase lifespan under multiple conditions of dietary restriction, such as lowering the concentration of bacteria fed to the worms in culture or eat-2 mutations, but not under conditions of lowered insulin signaling [10], making it a candidate for a transcriptional effector downstream of TOR signaling This working hypothesis was confirmed by the finding that PHA4 is required for the lifespan extension elicited by reduced LET-363/ TOR or RSKS-1/S6 kinase levels, both of which are independent of DAF-16 function [11]. Our findings yield novel insights about the regulation of growth by FKH, and how FKH and dFOXO are partially redundant in the regulation of d4E-BP expression
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