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 homeostasis. 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 haematopoiesis. 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 leukaemia. 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.

JAK signaling globally counteracts heterochromatic gene silencing

“…This interpretation is consistent with the fact that the chromatin-modifying proteins identified in this study were not found in the genome-wide RNAi screens based on changes in STAT92E transcriptional activity 13,14 . Moreover, mutations in the gene encoding NURF301, the large nucleosome remodeling factor (NURF) subunit, have been shown to cause blood tumors without affecting JAK/STAT activity 23 .…”

“…However, we found that reducing the dosage of Su (var)205, Su(var)3-9 and Rpd3 by half did not lead to increased levels of β-gal produced by GAS-lacZ (Fig. 2c,d), suggesting that the chromatin-modifying proteins HP1, Su(var)3-9 and Rpd3 do not normally repress JAK/STAT signaling.This interpretation is consistent with the fact that the chromatin-modifying proteins identified in this study were not found in the genome-wide RNAi screens based on changes in STAT92E transcriptional activity 13,14 . Moreover, mutations in the gene encoding NURF301, the large nucleosome remodeling factor (NURF) subunit, have been shown to cause blood tumors without affecting JAK/STAT activity 23 .…”

“…15). This effect is mediated by the JAK/STAT pathway, as reducing the dosage of Hop or STAT92E suppresses the largeeye phenotype 13,15 . We found that reducing or increasing HP1 levels significantly enhanced or suppressed the large-eye phenotype of GMR-upd flies, respectively (Fig.…”

“…These tumors manifest as melanotic masses of blood cell aggregates in the larval or adult body cavity, resulting from overproliferation and differentiation of particular blood cell types 10,11 . It has been shown that Tum-l induces hematopoietic tumors by overactivating STAT92E, as STAT92E is hyperphosphorylated by Hop Tum-l , and that reducing the gene dosage of STAT92E suppresses Tum-l tumorigenicity 5,6,13 . Classical genetic screens and genome-wide RNA interference (RNAi) analyses in D. melanogaster have led to the identification of many components involved in the JAK/STAT pathway or in its modulation 5,6,[13][14][15] .…”

Genomic RNAi screening in Drosophila S2 cells: what have we learned about host–pathogen interactions?

“…Two genome-wide screens have been performed to identify immune-associated components and mediators of the JAK/Stat signaling pathway using luciferase reporter systems [23,24] ( Table 1). Muller et al identified 67 positive regulators and 24 negative regulators of the pathway while Baeg et al identified 29 positive regulators and 92 negative regulators.…”