The ISWI-containing NURF complex regulates the output of the canonical Wingless pathway

Abstract

Wingless (Wg) signalling regulates the expression of its target genes through Pangolin, Armadillo and their interacting co-factors. In a genetic screen for Wg signalling components, we found that imitation switch (ISWI), a chromatin-remodelling ATPase, had a positive role in transducing the canonical Wg signal, promoting the expression of the Wg target senseless. ISWI is found in several chromatin-remodelling complexes, including nucleosome remodelling factor (NURF). The effect of interfering with the function of other components of the NURF complex in vivo mimics that of ISWI. The NURF complex is also required for the efficient expression of other Wg target genes. Armadillo interacts directly with the NURF complex in vitro and recruits it to Wg targets in cultured cells. Together, our results suggest that the ISWI-containing NURF complex functions as a co-activator of Armadillo to promote Wg-mediated transcription.

Figure 1

The activity of ISWI is crucial when Wingless signalling is compromised in the wing and eye of Drosophila. salE-Gal4 drove the expression of (A) UAS-GFP, (B) UAS-lgs^17E, (C) EP-iswi, (D) UAS-lgs^17E and EP-iswi, (E) UAS-iswi^K159R, and (F) UAS-lgs^17E and UAS-iswi^K159R. The arrow in (B) indicates the notched-wing phenotype caused by Lgs^17E. (G–L) The rough eye phenotype generated by sev-Wg was affected by ISWI activity. (G) GMR-Gal4/+, (H) GMR-Gal4/+; sev-wg/+, (I) GMR-Gal4/EP-iswi, (J) GMR-Gal4/EP-iswi; sev-wg/+, (K) GMR-Gal4/+; UAS-iswi^K159R/+, and (L) GMR-Gal4/+; UAS-iswi^K159R/sev-wg. It has been reported that universal or strong expression of ISWI^K159R can affect cell viability (Deuring et al, 2000). However, on expressing ISWI^K159R alone using salE-Gal4 or GMR-Gal4, we did not observe any wing or eye phenotypes (E,K). We believe that the lack of an obvious effect on viability is due to the moderate expression levels achieved with these drivers and possibly because of the timing of the expression. EP, enhancer P element; GFP, green fluorescent protein; ISWI, imitation switch; Lgs, legless; sev, sevenless; Wg, wingless; WT, wild type.

Figure 2

ISWI activity is required for sens expression in the wing disc. (A–C′″) Expression of UAS-iswi^K159R in the posterior compartment of the wing disc was controlled by en-Gal4, Gal80^ts (second-instar larvae were preserved at 29°C temperature for 2 days). Expression of (A–A″) Dll and (B) wg was not affected by ISWI^K159R. (C–C′″) The expression of sens, but not wg, was weakened by ISWI^K159R in the same wing disc. (D–D′″) The control disc expressing en-Gal4, Gal80^ts showed wild-type expression of sens and wg. (E–E′″) Expression of sens, but not wg, was weakened in iswi clones induced by hs-flp; FRT42 ubi-GFP Minute/FRT42: iswi¹. (F–F′″) The expression of sens and wg were unaltered in wild-type clones induced by hs-flp; FRT42 ubi-GFP Minute/FRT42 +. Dll, Distal-less; FRT, flippase recognition target; GFP, green fluorescent protein; hs-flp, heat shock-flippase; ISWI, imitation switch; sens, senseless; Wg, wingless; WT, wild type.

Figure 3

The NURF complex is required for sens expression in the wing disc. (A) The NURF complex comprises four subunits. (B–C′″) Expression of UAS-nurf301dsRNA in the posterior compartment of the wing disc by en-Gal4, Gal80^ts resulted in (B) a notched-wing phenotype in the posterior wing (arrow), and (C–C′″) weakening of sens expression without affecting wg expression in the posterior wing disc. (D–D′″) The expression of sens, but not wg, was weakened in nurf301 clones induced by hs-flp; ubi-GFP Minute FRT80/nurf301⁸ FRT80 (arrowhead). dsRNA, double-stranded RNA; GFP, green fluorescent protein; hs-flp, heat shock-flippase; NURF, nucleosome remodelling factor; sens, senseless; Wg, wingless.

Figure 4

The NURF complex is required for the transcriptional activation of some Wg targets in Kc cells. Cells were treated with the indicated dsRNAs for 4 days and total RNA were isolated for RT–PCR. The messenger RNA levels of actin, the ribosome protein L49 and TATA binding protein (TBP) were used as internal controls for normalization. Four Wg targets, (A) Nkd, (B) CG5895, (C) CG6234 and (D) Fz3 were measured for basal and activated transcription level. (E) The protein levels of ISWI and α-Tub were monitored in dsRNA-treated cells by Western blot. dsRNA, double-stranded RNA; Fz3, frizzled 3; GFP, green fluorescent protein; ISWI, imitation switch; Nkd, naked cuticle; NURF, nucleosome remodelling factor; RT–PCR, reverse transcription–PCR; Wg, wingless.

Figure 5

Recruitment of ISWI and the NURF complex by Armadillo. (A) Domain structure of full-length Armadillo and truncated Armadillo fusion constructs. (B) In vitro translated samples were pulled down by Armadillo constructs and detected by autoradiography. Lane 1, 10% input. Lane 2, pulled down by GST. Lane 3, pulled down by GST-ArmR1–8. Lane 4, pulled down by GST-ArmR1–10. Lane 5, pulled down by GST-ArmR11–C. (C) The NURF complex was pulled down by Armadillo constructs. HA-tagged ISWI and Myc-tagged NURF55 were detected by western blot. Lane 1, 20% input. Lane 2, pulled down by GST-ArmR1–8. Lane 3, pulled down by GST-ArmR1–10. Lane 4, pulled down by GST-ArmR11–C. Lane 5, pulled down by GST. (D) The binding of ISWI at the CG6234 locus. Kc cells were treated with control medium or Wg-conditioned medium (Wg-CM), combined with control RNAi, Arm RNAi or NURF301 RNAi. A locus containing Pan binding sites (C1) and a locus in the open reading frame (C0) were monitored for ISWI binding. ArmR, Armadillo repeat; ChIP, chromatin immunoprecipitation; GST, glutathione S-transferase; HA, haemagglutinin; ISWI, imitation switch; NURF, nucleosome remodelling factor; ORF, open reading frame; Pan, Pangolin; RNAi, RNA-mediated interference; Wg, wingless.