Genome wide RNAi screen identifies genes related to the insulin pathway as regulators of the transcriptional response to hypoxia

Andres Dekanty, Lazaro Centanin, Pablo Wappner. Fundación Instituto Leloir, Buenos Aires, ARGENTINA.

The transcriptional response to hypoxia is a highly conserved mechanism mediated by the heterodimeric ( α/ β) transcription factor HIF (hypoxia inducible factor). We have previously defined a hypoxia-responsive system homologous to HIF in Drosophila melanogaster, being the proteins Similar (Sima) and Tango (Tgo) the functional homologues of HIF- α and β subunits, respectively. Sima is regulated by oxygen through the gene fatiga (a prolyl hydroxylase) that behaves as an oxygen sensor. To gain insights on the regulation of the transcriptional response to hypoxia, a HIF-Responsive-Element (HRE) luciferase reporter was stably transfected in Drosophila S2 cells. Expression of the reporter is strongly induced in hypoxia or upon exposure to Deferoxamine (DFO), in a Sima/Tgo-dependent manner. The HRE-reporter was then used in a genome wide dsRNA-based screen. The screen led to the identification of novel genes presumably required for the transcriptional response to hypoxia, among which we identified components of the insulin-signaling pathway. We have demonstrated both in cell culture and in living embryos, that insulin is a potent activator of Sima-dependent transcription. This effect depends on PI3K and TOR pathways and involves accumulation of Sima protein as well as an increase of its nuclear localisation. It has been shown that PI3K and TOR pathways play a fundamental role in growth regulation. Increased activity promotes growth, while diminished signaling leads to cell and body size reduction. Interestingly, we found that fatiga loss-of-function led to body size reduction but fatiga sima double mutants display normal size. Consistent with this, Sima flip-out over-expression led to cell size reduction, strongly suggesting that Sima is a cell autonomous negative regulator of growth.

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