Long-range repression by multiple polycomb group (PcG) proteins targeted by fusion to a defined DNA-binding domain in Drosophila

A tethering assay was developed to study the effects or Polycomb group (PcG ) pro reins on gene expression In vivo. This system employed tile Su(Hw) DN A-binding domain (ZnF) to direct PcG proteins to transposons that carried t he white and yellow reporter genes. These reporters constituted naive senso rs of PcG effects, as bona fide PcG response elements (PREs) were absent fr om the constructs. To assess the effects of different genomic environments, reporter transposons integrated at nearly 40 chromosomal sites were analyz ed. Three PcG fusion proteins, ZnF-PC, ZnF-SCM, and ZnF-ESC, were studied, since biochemical analyses place these PcG proteins in distinct complexes. Tethered ZnF-PcG proteins repressed white and yellow expression at the majo rity of sites tested, with each fusion protein displaying a characteristic degree of silencing. Repression hy ZnF-PC was stronger than ZnF-SCM, which was stronger than ZnF-ESC, as judged by the percentage of insertion lines a ffected and the magnitude of the conferred repression. ZnF-PcC repression w as more effective at centric and telomeric reporter insertion sites, as com pared to euchromatic sites. ZnF-PcG proteins tethered as far as 3.0 kb away from the target promoter produced silencing, indicating that these effects were long range. Repression by ZnF-SCM required a protein interaction doma in, the SPM domain, which suggests tl-lat this domain is not primarily used to direct SCM to chromosomal loci. This targeting system is useful for stu dying protein domains and mechanisms involved in PcG repression in vivo.