Quantitative analysis of polycomb response elements (PREs) at identical genomic locations distinguishes contributions of PRE sequence and genomic environment

We adapted the ΦC31 site-specific integration tool to enable systematic quantitative comparison of PREs and sequence variants at identical genomic locations. In this adaptation, a miniwhite (mw) reporter in combination with eye-pigment analysis gives a quantitative readout of PRE function. We compared the Hox PRE Frontabdominal-7 (Fab-7) with a PRE from the vestigial (vg) gene at four landing sites. The analysis revealed that the Fab-7 and vg PREs have fundamentally different properties, both in terms of their interaction with the genomic environment at each site and their inherent silencing abilities. Furthermore, we used the ΦC31 tool to examine the effect of deletions and mutations in the vg PRE, identifying a 106 bp region containing a previously predicted motif (GTGT) that is essential for silencing.This analysis showed that different PREs have quantifiably different properties, and that changes in as few as four base pairs have profound effects on PRE function, thus illustrating the power and sensitivity of ΦC31 site-specific integration as a tool for the rapid and quantitative dissection of elements of PRE design.Polycomb/Trithorax response elements (PREs) are cis-regulatory DNA elements that recruit both the Polycomb group (PcG) and Trithorax group (TrxG) proteins, required respectively for gene silencing and activation [1-3]. PREs were first identified in the homeotic gene clusters of the Bithorax complex (BX-C) [4-6] and the Antennapedia complex (ANT-C) [7] in Drosophila. Genomewide studies in flies and vertebrates have since identified several hundred additional PcG target genes involved in cell-fate specification, cell signaling and proliferation [8-18]. However, functional studies of PRE elements themselves have been performed for only a few of these loci. These studies, based on transgenic reporter assays, have shown that several Drosophila PREs share common molecular and genetic features. These include recruitment of PcG and TrxG proteins to an ectopi