Context dependent role of Pho binding sites in Polycomb complex recruitment in Drosophila

Polycomb group (PcG) proteins maintain the silenced state of key developmental genes, but how these proteins are recruited to specific regions of the genome is still not completely understood. In Drosophila, PcG proteins are recruited to Polycomb response elements (PREs) comprised of a flexible array of sites for sequence-specific DNA binding proteins, “PcG recruiters”, including Pho, Spps, Cg, GAF and many others. Pho is thought to play a central role in PcG recruitment. Early data showed that mutation of Pho binding sites in PREs in transgenes abrogated the ability of those PREs to repress gene expression. In contrast, genome-wide experiments in pho mutants or by Pho knockdown showed that PcG proteins can bind to PREs in the absence of Pho. Here we directly addressed the importance of Pho binding sites in two engrailed (en) PREs at the endogenous locus and in transgenes. Our results show that Pho binding sites are required for PRE activity in transgenes with a single PRE. In a transgene, two PREs together lead to stronger, more stable repression and confer some resistance to the loss of Pho binding sites. Making the same mutation in Pho binding sites has little effect on PcG-protein binding at the endogenous en gene. Overall, our data support the model that Pho is important for PcG binding but emphasize how multiple PREs and chromatin environment increase the ability of PREs to function in the absence of Pho. Overall design: Genome-wide binding of different PcG recruiters or PcG proteins (Pho, Spps, Ph, Ez, Sfmbt) in Crispr generated lines in an en?1.5 background. 1.5kb fragments carrying either wild-type PRE1 and PRE2 of the engrailed gene or MutPRE2 (WT PRE1 and Pho site mutations in PRE2) or Mut PRE1+2 (Pho site mutations in both PRE1 and PRE2). These lines were generated specifically to look at PcG recruitment to the engrailed PREs. H3K27me3 ChIP seq was carried out on transgenic lines with the split mini-white transgene constuct (inserted at 2L:9437482 (Dm version 6)) with either no insert (control), a WT line carrying WT enPRE1+2, PhoKO PRE1+2) or MutallGAGA PRE1+2. These experiments were carried out to look at the levels of H3K27me3 recruited over the reporter genes and genes neighboring the insertion site to see the impact of H3K27me3 in the absence of Pho binding sites or GAGA binding sites.