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

Helena Okulski,Leonie Ringrose,Sheetal Bhalerao,Birgit Druck

doi:10.1186/1756-8935-4-4

Abstract

BackgroundPolycomb/Trithorax response elements (PREs) are cis-regulatory elements essential for the regulation of several hundred developmentally important genes. However, the precise sequence requirements for PRE function are not fully understood, and it is also unclear whether these elements all function in a similar manner. Drosophila PRE reporter assays typically rely on random integration by P-element insertion, but PREs are extremely sensitive to genomic position.ResultsWe 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.ConclusionsThis 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.

Highlights

Polycomb/Trithorax response elements (PREs) are cis-regulatory elements essential for the regulation of several hundred developmentally important genes
These motifs alone are not sufficient to predict all PREs in the genome, nor to fulfill PRE function [37], and functional roles for additional DNA sequence motifs have been defined for specific PREs, including binding sites for Dorsal switch protein (DSP)1 [37], Grainyhead (GRH) [38], the SP1/Kruppel-like factor (KLF) family of transcription factors [39] and several other unidentified proteins [1,2,25]
The mw gene is typically used as a sensitive reporter gene for PRE effects [5], and serves here both as a transgenic marker and as a reporter of PRE activity

Summary

Introduction

Polycomb/Trithorax response elements (PREs) are cis-regulatory elements essential for the regulation of several hundred developmentally important genes. Clusters of pairs of these motifs are sufficient for computational detection of a subset of known Drosophila PREs and for the prediction of further PREs, many of which have been confirmed experimentally [8,36] These motifs alone are not sufficient to predict all PREs in the genome, nor to fulfill PRE function [37], and functional roles for additional DNA sequence motifs have been defined for specific PREs, including binding sites for Dorsal switch protein (DSP)1 [37], Grainyhead (GRH) [38], the SP1/Kruppel-like factor (KLF) family of transcription factors [39] and several other unidentified proteins [1,2,25]. With the use of sequence mining, further DNA motifs have been found to be enriched at PRE elements [8,11], and have been proposed to play a role in PRE regulation, but their function has not been tested experimentally

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