The nucleosome remodeling factor ISWI functionally interacts with an evolutionarily conserved network of cellular factors

Abstract

ISWI is an evolutionarily conserved ATP-dependent chromatin remodeling factor playing central roles in DNA replication, RNA transcription, and chromosome organization. The variety of biological functions dependent on ISWI suggests that its activity could be highly regulated. Our group has previously isolated and characterized new cellular activities that positively regulate ISWI in Drosophila melanogaster. To identify factors that antagonize ISWI activity we developed a novel in vivo eye-based assay to screen for genetic suppressors of ISWI. Our screen revealed that ISWI interacts with an evolutionarily conserved network of cellular and nuclear factors that escaped previous genetic and biochemical analyses.

Figure 1.—

Loss of ISWI function by the EGUF approach causes specific eye phenotypes. Drosophila adult eyes obtained with the EGUF mitotic clonal approach (Stowers and Schwarz 1999) bear in homozygosis a wild-type 2R chromosome (A), an ISWI² allele (B), an ISWI² allele in the presence of one extra copy of the wild-type ISWI⁺ gene (C), or a copy of ISWI^K159R defective in its ATPase activity (D), a brm² (E) or kis¹ allele (F), an ISWI² allele in the presence of one copy of acf1[1] (G), or E(bx)^Nurf301-2 (H) alleles. The white arrowhead indicates the eye color variegation, while the black arrowhead indicates loss of cell identity defects in which bristles grow in eye territories normally occupied by photoreceptors. The ISWI-EGUF eye phenotype is caused by the progressive depletion, during eye development, of the ISWI mRNA/protein pool present before mitotic recombination in ISWI² heterozygous mother cells. As a consequence, ISWI progressive loss of activity during eye development, occurring after mitotic recombination, could be accelerated by loss of factors positively regulating its activity, like acf1 and E(bx), thus explaining the enhancement of ISWI-EGUF eye defects we observe. On the other hand, the presence of an extra copy of the wild-type ISWI⁺ gene can complement eye defects caused by the recombination-dependent ISWI genetic loss.

Figure 2.—

ISWI-EGUF genetic screen strategy. (A) The use of distinguishable fluorescent transformation markers in the adult eyes allows us to follow the mutator (YFP) and the jumpstarter (CFP) elements independently. (B and C) Crossing scheme for the identification of new PiggyBac mutator (B) and third chromosome EP insertions (C) dominantly modifying ISWI-EGUF eye phenotypes. For both the PiggyBac mutator and the EP screen, in the absence of mitotic recombination, the presence of the GMR-hid transgene generates adult eyes without ommatidia but slightly pigmented (Stowers and Schwarz 1999), allowing their unambiguous distinction from the mitotic recombinant experimental class. The presence of w⁺-marked EP insertions allowed the scoring of the mitotic recombinant experimental class in the EP screen. Mut, mutator; Jump, jumpstarter.

Figure 3.—

ISWI genetically interacts with a wide range of cellular components. The 99 potential protein-coding loci corresponding to ISWI-EGUF dominant modifiers are clustered in concentric circles as nodes, colored according to their interaction class (A), current gene ontology (GO) categories, as indicated in the key (B), and their intersection with ISWI interactors isolated in the ISWI^K159R screen (Burgio et al. 2008) (C). The edges represent known physical and genetic interactions identified with the experimental system indicated in the key.

Figure 4.—

Loss of ISWI function in the eye can be dominantly suppressed by mutations in mbf1, ttk, eff, and wun. (A) Eye phenotypes resulting from an eye homozygous for ISWI² (ISWI-EGUF eye) carrying an EP insertion mapping the mbf1, ttk, and eff genes or a PiggyBac insertion in the wun gene. (B) To validate the genetic interactions we scored, other alleles mapping mbf1, ttk, eff, and wun were tested in the ISWI-EGUF eye assay.

Figure 5.—

Evolutionarily conserved network of regulation of ISWI. BioGrid analysis is shown of known genetic and physical interactions existing between the genes identified in the fly “neuronal morphogenesis” and the worm “multiple cell fate” screens and the modifier genes we picked in both the ISWI-EGUF and the ISWI^K159R eye screens (Andersen et al. 2006; Parrish et al. 2006; Burgio et al. 2008). Our analysis predicted (A) 1 big and (B) 12 small interaction networks comprising ISWI genetic interactors isolated in the four different genetic screens analyzed, linked by “connecting” nodes corresponding to interactors not isolated in previous ISWI related screens. The big network comprises 93 nodes while the 12 small networks are constituted of 33 original nodes and 41 connecting new nodes. (C) Alleles of genes corresponding to 63% of the neuronal morphogenesis, 90% of the multiple cell fate, and 50% of the connecting nodes genetically interacted with the ISWI-EGUF or the ISWI^K159R eye assays. These interaction frequencies are much greater than the frequencies of ISWI interaction we normally get with eye-based screens (usually in the ∼1–11% range), suggesting that the gene network analysis we conducted increased our ability to predict ISWI interactors. The edges represent known physical and genetic interactions identified with the experimental system indicated in the key. ISWI-EGUF or ISWI^K159R interacting genes are highlighted in boldface type and have a bigger node size.

Figure 6.—

Cytofluorimetric analysis of ISWI mutant cells. Cell populations derived ex vivo from wild-type and ISWI mutant neuroblasts (A) and total imaginal (B) and eye-antennal imaginal (C) discs were analyzed by flow cytometry. ISWI imaginal disc cells showed a significant decrease of G₁ and G₂/M peaks (red arrows) and an increase in the pre-G₁ peak (purple arrow). On the other hand, when compared to their wild-type counterpart, ISWI mutant neuroblasts showed a small but reproducible increase in the G₂/M peak (green arrow) and a faster S phase (blue arrow). These cell cycle defects can be in part or completely suppressed by some of the Su(ISWI)'s we isolated.