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Identification of JAK/STAT signalling components by genome-wide RNA interference

Abstract

Signalling pathways mediating the transduction of information between cells are essential for development, cellular differentiation and homeostasis1. Their dysregulation is also frequently associated with human malignancies. The Janus tyrosine kinase/signal transducer and activator of transcription (JAK/STAT) pathway represents one such signalling cascade whose evolutionarily conserved roles include cell proliferation and haematopoiesis2. Here we describe a systematic genome-wide survey for genes required for JAK/STAT pathway activity. Analysis of 20,026 RNA interference (RNAi)-induced phenotypes in cultured Drosophila melanogaster haemocyte-like cells identified interacting genes encoding 4 known and 86 previously uncharacterized proteins. Subsequently, cell-based epistasis experiments were used to classify these proteins on the basis of their interaction with known components of the signalling cascade. In addition to multiple human disease gene homologues, we have found the tyrosine phosphatase Ptp61F and the Drosophila homologue of BRWD3, a bromo-domain-containing protein disrupted in leukaemia3. Moreover, in vivo analysis demonstrates that disrupted dBRWD3 and overexpressed Ptp61F function as suppressors of leukaemia-like blood cell tumours. This screen represents a comprehensive identification of novel loci required for JAK/STAT signalling and provides molecular insights into an important pathway relevant for human cancer. Human homologues of identified pathway modifiers may constitute targets for therapeutic interventions.

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Figure 1: Genome-wide RNAi screen for JAK/STAT signalling factors.
Figure 2: Analysis of JAK/STAT activity modulators.
Figure 3: dBRWD3 functions as a JAK/STAT pathway component.
Figure 4: Ptp61F is a tumour suppressor in vivo.

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Acknowledgements

We are grateful to R. Paro for providing reagents to establish the genome-wide RNAi library. We wish to thank M. Yamayuchi and P. Karsten for the original 2 × DrafSTAT(wt) and Socs36E promoter plasmids, C. Hunter for the sid-1 plasmid, K. Bartscherer for advice and help with tissue culture experiments, T. Horn for bioinformatics support, and M. Stricker, B. Mosterman, I. Plischke and S. Häder for technical help. We thank H. Jäckle, S. Cohen, M. Osborn, R. Paro and N. Pelte for comments on the manuscript. P.M. was supported by a fellowship from the German National Academic Foundation (Studienstiftung). Research in M.B. and M.P.Z. laboratories was supported in part by Emmy-Noether grants from the Deutsche Forschungsgemeinschaft.Author Contributions This work has been a collaborative effort between the groups of M.B. and M.P.Z.; the Boutros laboratory contributing the functional genomic expertise to dissect signalling pathways and the Zeidler laboratory expertise working with JAK/STAT signalling.

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Correspondence to Martin P. Zeidler or Michael Boutros.

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Reprints and permissions information is available at npg.nature.com/reprintsandpermissions. The authors declare no competing financial interests.

Supplementary information

Supplementary Methods

Description of Supplementary Methods including: constructs and pathway reporter; genome-wide RNAi library; high-throughput RNAi screening; computational analysis; Sequence analysis; epistasis experiments and Ptp61F phenotype in cells; and genetics This file also contains additional references and Supplementary Figure Legends. (DOC 196 kb)

Supplementary Tables

Supplementary Table S1–S7. (PDF 428 kb)

Supplementary Figure S1

Overview of primary RNAi screen data. (PDF 1361 kb)

Supplementary Figure S2

Loss of JAK/STAT pathway components and hopTuml induced tumour formation. (PDF 451 kb)

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Müller, P., Kuttenkeuler, D., Gesellchen, V. et al. Identification of JAK/STAT signalling components by genome-wide RNA interference. Nature 436, 871–875 (2005). https://doi.org/10.1038/nature03869

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