Genetic Identification of a Network of Factors that Functionally Interact with the Nucleosome Remodeling ATPase ISWI

Abstract

Nucleosome remodeling and covalent modifications of histones play fundamental roles in chromatin structure and function. However, much remains to be learned about how the action of ATP-dependent chromatin remodeling factors and histone-modifying enzymes is coordinated to modulate chromatin organization and transcription. The evolutionarily conserved ATP-dependent chromatin-remodeling factor ISWI plays essential roles in chromosome organization, DNA replication, and transcription regulation. To gain insight into regulation and mechanism of action of ISWI, we conducted an unbiased genetic screen to identify factors with which it interacts in vivo. We found that ISWI interacts with a network of factors that escaped detection in previous biochemical analyses, including the Sin3A gene. The Sin3A protein and the histone deacetylase Rpd3 are part of a conserved histone deacetylase complex involved in transcriptional repression. ISWI and the Sin3A/Rpd3 complex co-localize at specific chromosome domains. Loss of ISWI activity causes a reduction in the binding of the Sin3A/Rpd3 complex to chromatin. Biochemical analysis showed that the ISWI physically interacts with the histone deacetylase activity of the Sin3A/Rpd3 complex. Consistent with these findings, the acetylation of histone H4 is altered when ISWI activity is perturbed in vivo. These findings suggest that ISWI associates with the Sin3A/Rpd3 complex to support its function in vivo.

The Nucleosome Remodeling Factor ISWI Functionally Interacts With an Evolutionarily Conserved Network of Cellular Factors

“…Some ISWI-EGUF modifiers (i.e., mbf1, ttk, eff, stg, and cpo) were validated by testing other alleles of these genes, available from public stock centers, using the genetic screen scheme used for testing EP insertions. Mutations in genes corresponding to ''neuronal morphogenesis,'' ''multiple cell fate,'' and ''connecting'' nodes were obtained from public stock centers and tested for their ability to interact in the ISWI-EGUF eye assay, using the genetic screen scheme used for testing EP insertions, and in the ISWI K159R eye assay according to Burgio et al (2008). Bioinformatic and cell cycle analyses: For the BioGrid analysis (Breitkreutz et al 2008), each En(ISWI), Su(ISWI), and Bi(ISWI) ISWI-EGUF interaction was associated with a single gene on the basis of the insertion DNA sequence data available in FlyBase (www.flybase.org) and the iPCR analysis we conducted on the PiggyBac insertions.…”

“…and genes encoding for chromatin covalent modifiers, established that eye-based genetic screens in flies could be a powerful tool for the in vivo dissection of chromatinremodeling signaling pathways occurring in the nucleus Armstrong et al 2005;Burgio et al 2008;Sala and Corona 2009). Moreover, the ISWI K159R screen established that ISWI function could be modulated in vivo by a variety of cellular factors that have escaped previous biochemical analyses .…”

“…1 ISWI genetic interactors in the higher eukaryote Drosophila melanogaster . Loss of ISWI function, by eye-specific misexpression of the dominant negative allele ISWI K159R , produces catalytically inactive ISWI that is incorporated into native complexes, giving rise to rough and reduced eye phenotypes in otherwise healthy flies (Deuring et al 2000;Burgio et al 2008). In a previous study, we used this in vivo eye assay to conduct an unbiased genetic screen for mutations in genes that dominantly modify phenotypes resulting from the misexpression of ISWI K159R in the eye .…”

Functional antagonism between histone H3K4 demethylases in vivo

“…For the ISWI K159R interaction test, yw; eyGAL4; UAS-ISWI K159R / T(2:3) virgins were crossed with the dLsd1 DN /TM3. The F1 progeny that did not carry any balancer were scored based on the severity of eye phenotype, as in Burgio et al (2008). The effect of dLsd1 and Lid mutations on white variegation was studied by crossing females homozygous for the In(1)w m4h allele with males heterozygous for dLsd1 and Lid mutations.…”

Regulation of ISWI chromatin remodelling activity

“…ISWI-PARP interaction was detected for the first time in an unbiased genetic screening aimed at the identification of factors modifying phenotypes caused by loss of ISWI function in flies. This screening provided the first genetic interaction map of potential regulators of ISWI in the higher eukaryote Drosophila melanogaster (Arancio et al 2010;Burgio et al 2008). Poly-ADP-ribosylated dISWI displays a reduction in both nucleosome binding affinity as well as ATPase activity (Fig.…”