Abstract
Apoptosis is a conserved form of programmed cell death firmly established in the aetiology, pathogenesis and treatment of many human diseases. Central to the core machinery of apoptosis are the caspases and their proximal regulators. Current models for caspase control involve a balance of opposing elements, with variable contributions from positive and negative regulators among different cell types and species1. To advance a comprehensive view of components that support caspase-dependent cell death, we conducted a genome-wide silencing screen in the Drosophila model. Our strategy used a library of double-stranded RNAs together with a chemical antagonist of Inhibitor of apoptosis proteins (IAPs) that simulates the action of native regulators in the Reaper and Smac (also known as Diablo) families2. Here we present a highly validated set of targets that is necessary for death provoked by several stimuli. Among these, Tango7 is identified as a new effector. Cells depleted for this gene resisted apoptosis at a step before the induction of effector caspase activity, and the directed silencing of Tango7 in Drosophila prevented caspase-dependent programmed cell death. Unlike known apoptosis regulators in this model system3, Tango7 activity did not influence stimulus-dependent loss of Drosophila DIAP1 (also known as th and IAP1), but instead regulated levels of the apical caspase Dronc (Nc). Similarly, the human Tango7 counterpart, PCID1 (also known as EIF3M), impinged on caspase 9, revealing a new regulatory axis affecting the apoptosome.
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Acknowledgements
We thank X. Tu, L. Durham, L. Lum, P. Beachy, M. Roth, X. Wang, P. Harran, Y. Lazebnik, D. Dorris, the University of Texas Southwestern Medical Center (UTSW) High Throughput Screening laboratory and the Live Cell Imaging facility for technical and material support; Bloomington Stock Center, K. Basler, National Institute of Genetics (NIG) stock center (Japan) for fly lines; K. White, B. Hay and the Hybridoma Bank for antibodies; V. Malhotra for the pMT-Tango7 plasmid; J. Seeman and the Wang laboratory for discussions; and M. Allen for administrative support. This work was supported by grants from the National Institute of General Medical Sciences, the National Institute on Alcohol Abuse and Alcoholism and the UTSW High Impact/High Risk Grant Program. N.L. is supported by a National Research Service Award.
Author Contributions S.K.C., N.L., P.C., K.A.G. and K.P. planned and performed experiments and analysed results. All authors discussed results. J.M.A. planned experiments, analysed results and wrote the paper together with S.K.C., aided by contributions from N.L. and P.C.
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Supplementary Information
This file contains Supplementary Figure 1 with Legend and Supplementary Tables 1-3. (PDF 398 kb)
Supplementary Table
This file contains Supplementary Table 4 which shows dsRNA Library Sequences. (XLS 9605 kb)
Supplementary Movie 1
This movie shows S2R+ cells treated with Smac-mimetic compound to induce apoptosis. Time lapse covers period of 12 hrs. (MOV 4144 kb)
Supplementary Movie 2
This movie shows S2R+ cells treated with UV irradiation and control AmpR dsRNA. Time lapse covers period of 22 hrs. (MOV 7758 kb)
Supplementary Movie 3
This movie shows S2R+ cells treated with UV irradiation and Dronc dsRNA. Time lapse covers period of 22 hrs. (MOV 5859 kb)
Supplementary Movie 4
This movie shows S2R+ cells treated with UV irradiation and Tango7 dsRNA. Time lapse covers period of 22 hrs. (MOV 9419 kb)
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Chew, S., Chen, P., Link, N. et al. Genome-wide silencing in Drosophila captures conserved apoptotic effectors. Nature 460, 123–127 (2009). https://doi.org/10.1038/nature08087
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DOI: https://doi.org/10.1038/nature08087
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