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.

Figure 1. Primary screen for genes required for HIF–dependent transcription.

(A) S2-HRE-Luc cells treated with dsRNA against gfp (negative control), sima or tango were exposed or not to DFO. Luciferase induction by DFO was abrogated in cells depleted from sima or tango. (B) Scatter plot of the average Z-score (see Materials and Methods) of the whole set of data of the primary screen. dsRNAs which reduced reporter gene expression with a Z-score of less than −2.5 (cut-off line) were selected as positive hits of the primary screen for further analysis.

Figure 2. Argonaute 1 (Ago1) and the miRNA machinery are necessary for adaptation to hypoxia.

(A) Western blot showing Ago1 strong reduction in cells treated with dsRNA against ago1 during 4 days. Two different dsRNAs, ago 1.1 and ago 1.2 were used with identical results. Extracts from control cells were loaded at different amounts. Remaining Ago1 protein levels were 10% relative to controls after 4 days of RNAi treatment. Hsp70 was used as a loading control. (B) mRNA levels of two different HIF target genes, fatiga and ldh, were analyzed by real time PCR in cells exposed to hypoxia (1% O₂) for 16 hours in comparison to those of cells maintained in normoxia. sima or ago1 dsRNAs largely prevented hypoxic induction of ldh and fatiga transcripts. (C) S2-HRE-Luc cells were treated with dsRNA against gfp, ago1, dicer-1, drosha or gw182 and then exposed to DFO or 1% O₂. Whereas the gfp dsRNA had no effect on luciferase induction, silencing of any of the other genes strongly reduced luciferase induction by DFO or hypoxia. Data are represented as fold induction respect to control cells treated with dsRNA against gfp, and maintained in normoxia. (D) Analysis of the proportion of cells in apoptosis revealed that cells treated with ago1 dsRNA were as sensitive to hypoxia as cells treated with sima dsRNA, whereas untreated cells or cells treated with ago2 dsRNA were remarkably more resistant to low oxygen. After exposure to hypoxia, cells were stained with propidium iodide (PI) and Hoescht, and observed under a fluorescence microscope. The proportion of dying cells (PI positive) was determined using the CellProfiler cell image analysis software (Chi² test *p<0.05; ***p<0.001). (E–F) Transgenic embryos bearing the hypoxia inducible reporter LDH-lacZ were exposed to hypoxia (3% O₂) during 4 hours, and reporter gene activity was analyzed by X-gal staining (E) or quantitative β-galactosidase assays (F). The transgenic reporter is silent in normoxic wild type individuals, and strongly induced upon exposure to hypoxia (E). In ago1^k08121 homozygous mutants the expression of the reporter in hypoxia is clearly reduced (E–F; p<0.01). N = Normoxia; H = Hypoxia.

Figure 3. Hypoxic accumulation of Sima protein and mRNA is prevented in cells treated with ago1 dsRNA.

(A) Anti-Sima western blot analysis reveals that hypoxic accumulation of Sima is reduced in ago1 RNAi treated cells (24 h at 1% O₂). (B) Real time PCR revealed that sima mRNA is strongly induced in cells exposed to hypoxia, and this induction is largely prevented in cells treated with ago1 or GW182 dsRNA.

Figure 4. PBs accumulate in cells exposed to hypoxia in an Ago1- and GW182-dependent manner.

(A) S2R+ cells were maintained in normoxia or exposed to hypoxia (1% O₂) for different time periods, then fixed and stained with an anti-DCP1 or anti-Hedls antibodies, two PBs specific markers. The PB area per cell was determined, revealing that PBs accumulate in a transient manner in cells exposed to hypoxia, peaking at 6 h after the onset of the hypoxic treatment, and decreasing at 8 h (one-way ANOVA and Dunnett multiple comparison post-Test, **p<0.01). (B) ago1 or GW182 dsRNA treatment affect PB basal levels and prevent PB accumulation upon exposure of the cells to 1% O₂ for 6 h. (one-way Anova and SNK multiple comparisons post-test, p<0.01).

Figure 5. Model for sima regulation by the miRNA machinery.

An unknown (“X”) factor that directly or indirectly inhibits sima transcription is silenced by the miRNA machinery. When cells are depleted from Ago1, the factor X accumulates thereby preventing sima transcriptional induction in hypoxia.