The Drosophila nucleosome remodeling factor NURF is required for Ecdysteroid signaling and metamorphosis

Abstract

Drosophila NURF is an ISWI-containing ATP-dependent chromatin remodeling complex that regulates transcription by catalyzing nucleosome sliding. To determine in vivo gene targets of NURF, we performed whole genome expression analysis on mutants lacking the NURF-specific subunit NURF301. Strikingly, a large set of ecdysone-responsive targets is included among several hundred NURF-regulated genes. Null Nurf301 mutants do not undergo larval to pupal metamorphosis, and also enhance dominant-negative mutations in ecdysone receptor. Moreover, purified NURF binds EcR in an ecdysone-dependent manner, suggesting it is a direct effector of nuclear receptor activity. The conservation of NURF in mammals has broad implications for steroid signaling.

Figure 1.

Analysis of Nurf301 mutants. (A) Domain structure of NURF301 and its human homolog BPTF. The location of EMS-induced lesions in 12 Nurf301 mutants is shown. (B) Pupariation frequency of hemizygous Nurf301 mutant animals. Null alleles of Nurf301 fail to pupariate.

Figure 2.

Whole genome expression analysis of Nurf301 mutants. (A) Gene ontology (GEO) classification of genes decreased in expression in Nurf301 mutants. Potential ecdysone target genes are bracketed. (B) Expression of known ecdysone target genes in Nurf301 mutants relative to wild type. Color bar denotes the magnitude and direction of change. Full microarray data sets are available at http://home.ccr.cancer.gov/badenhorst.

Figure 3.

NURF mutants do not express EcR target genes. Northern analysis of Sgs1, Sgs3, and Eig71Ee expression (A) and RT-PCR analysis of Eig71Ea, ImpE2, and Fbp1 expression (B) in Nurf301 and Iswi mutants relative to rp49. For brevity, the heteroallelic Nurf301 mutant combinations Nurf301²/Nurf301³ and Nurf301²/Nurf301¹² are denoted by Nurf301^2/3 and Nurf301^2/12. The Iswi¹/Iswi² mutant combination is denoted by Iswi^1/2. Using the null Nurf301²/Nurf301³ allelic combination in the Northern and RT-PCR analyses to validate microarray data (obtained using the null Nurf301²/Nurf301⁸ allelic combination) provides assurance against the potential influence of second site mutations in these strains. In B, data from two independent samples for each genotype are shown. (C) Expression of an Sgs3-GFP reporter transgene (Biyasheva et al. 2001) in Sgs3-GFP; Nurf301²/Nurf301² homozygous mutants (-/-) and Sgs3-GFP; Nurf301²/TM6B, Tb¹ siblings (-/+). Arrowheads label salivary glands.

Figure 4.

NURF binds EcR in an ecdysone-dependent manner. (A) Structure of EcR isoforms and derivatives tested for NURF binding. (A/B) Variable-length N-terminal domain; (C) DNA-binding domain; (E) ligand-binding domain; (F) variable-length C-terminal domain; (GAL4 DBD) GAL4 DNA-binding domain; (AF1) activation function 1; (AF2) activation function 2 (amino acids 636-655 of the LBD). Full details of the GAL4 DBD-EcR fusions used in NURF pull-down are listed in the Supplemental Material. (B) Purified Flag-tagged recombinant NURF binds in vitro-translated EcR-A and EcR-B2 isoforms, and GBT-EL but not GBT-EL650 or GBT-EL/F645A. Binding is only detected in the presence (+) of 10 μM 20-hydroxyecdysone. Addition of solvent, ethanol, alone (-) cannot induce binding of NURF to EcR isoforms.

Figure 5.

NURF mutants enhance dominant-negative EcR phenotypes. Embryos derived from females overexpressing a dominant-negative EcR variant (UAS-EcR-F645A) under the control of slbo-GAL4 show a low frequency (23%) of abnormal dorsal appendages. Removing one copy of a NURF subunit (by introducing either a Nurf301, Iswi, or Nurf38 mutant allele into the slbo-GAL4::UAS-EcR-F645A background) increases the frequency and severity of these abnormalities. (A, left) Dorsal appendages of a wild-type egg. Dorsal appendages of embryos from GAL4::UAS-EcR-F645A; Nurf301/+ females show malformation (arrowheads) and fusion (arrows). (B) Frequency of abnormal dorsal appendages in embryos from slbo-GAL4::UAS-EcR-F645A females and slbo-GAL4::UAS-EcR-F645A females containing one copy of either a Nurf301, Iswi, or Nurf38 mutant allele. (n) The number of embryos scored.