Drosophila Genome-Wide RNAi Screen Identifies Multiple Regulators of HIF–Dependent Transcription in Hypoxia

Abstract

Hypoxia-inducible factors (HIFs) are a family of evolutionary conserved alpha-beta heterodimeric transcription factors that induce a wide range of genes in response to low oxygen tension. Molecular mechanisms that mediate oxygen-dependent HIF regulation operate at the level of the alpha subunit, controlling protein stability, subcellular localization, and transcriptional coactivator recruitment. We have conducted an unbiased genome-wide RNA interference (RNAi) screen in Drosophila cells aimed to the identification of genes required for HIF activity. After 3 rounds of selection, 30 genes emerged as critical HIF regulators in hypoxia, most of which had not been previously associated with HIF biology. The list of genes includes components of chromatin remodeling complexes, transcription elongation factors, and translational regulators. One remarkable hit was the argonaute 1 (ago1) gene, a central element of the microRNA (miRNA) translational silencing machinery. Further studies confirmed the physiological role of the miRNA machinery in HIF–dependent transcription. This study reveals the occurrence of novel mechanisms of HIF regulation, which might contribute to developing novel strategies for therapeutic intervention of HIF–related pathologies, including heart attack, cancer, and stroke.

Genome-scale RNAi on living-cell microarrays identifies novel regulators of Drosophila melanogaster TORC1–S6K pathway signaling

“…Several lines of evidence (for review, see Wouters and Koritzinsky 2008) suggest that under hypoxic conditions, decreased TORC1 signaling inhibits growth via HIF-1; accordingly, Tor, raptor, and Pk61C/PDK1 all scored in an RNAi screen for inhibitors of the HIF transcriptional response (Dekanty et al 2010). Beyond these canonical TORC1 pathway members, the 66 hits from the HIF-dependent-transcription screen included eight of our low-pS6 hits (>30-fold enrichment) (Fig.…”

“…6) demonstrate a conserved role for the Argonaute-GW complex in regulation of the TORC1 pathway. This relationship is underscored by the results of other growth-related RNAi screens in which AGO1 and raptor Dekanty et al 2010) or gw and raptor (Bjorklund et al 2006; primary screen of Friedman and Perrimon 2006) share a phenotype. It will be interesting to tease apart the relationship between the AGO-GW and TORC1 pathways.…”

“…We performed a meta-analysis comparing hits between the present study and several other cell-growth-related Drosophila RNAi screens (Kiger et al 2003;Bettencourt-Dias et al 2004;Boutros et al 2004;Bjorklund et al 2006;Friedman and Perrimon 2006;Goshima et al 2007;Sepp et al 2008;Mummery-Widmer et al 2009;Ravi et al 2009;Sims et al 2009;Dekanty et al 2010;Kockel et al 2010). We also examined prior Drosophila genome-scale RNAi screens whose measured phenotypes were not explicitly growthrelated but whose hits included one or more canonical TORC1 pathway members (Agaisse et al 2005;Guo et al 2008;Zhou et al 2008;Zhang et al 2010).…”

“…7E)-by Western blots, and all seven caused reductions in either dS6K phosphorylation or dS6K levels. It is possible that these noncanonical HIF-pathway regulators in the Dekanty et al (2010) screen exert their effects on HIF-dependent transcription by modulating the TORC1-S6K axis.…”

The Hypoxia-Inducible Factor-1α in Angiogenesis and Cancer: Insights from the Drosophila Model

“…The ago1 (argonaute 1) gene, which has a critical role in microRNA silencing processes, was also identified in this screen as a regulator of Sima in hypoxia. Given the involvement of Ago1 in Sima regulation, the authors went further to show that the microRNA pathway has a central role in HIF-dependent transcription, and also that Sima mRNA stabilization has a critical role in the Drosophila response to hypoxia [183].Further work on Sima regulators has uncovered several modifiers of Sima function in hypoxia in Drosophila, such as the microRNA miR-190, the TIP60 chromatin remodeling complex and the RNA-binding protein Musashi [184][185][186] (Figure 2). miR-190 acts as a positive regulator of the hypoxic response by targeting directly the propyl-4-hydroxylase Fga, which is the principal negative regulator of Sima.…”