Abstract
BackgroundChromatin boundaries, also known as insulators, regulate gene activity by organizing active and repressive chromatin domains and modulate enhancer-promoter interactions. However, the mechanisms of boundary action are poorly understood, in part due to our limited knowledge about insulator proteins, and a shortage of standard assays by which diverse boundaries could be compared.ResultsWe report here the development of an enhancer-blocking assay for studying insulator activity in Drosophila cultured cells. We show that the activities of diverse Drosophila insulators including suHw, SF1, SF1b, Fab7 and Fab8 are supported in these cells. We further show that double stranded RNA (dsRNA)-mediated knockdown of SuHw and dCTCF factors disrupts the enhancer-blocking function of suHw and Fab8, respectively, thereby establishing the effectiveness of using RNA interference in our cell-based assay for probing insulator function.ConclusionThe novel boundary assay provides a quantitative and efficient method for analyzing insulator mechanism and can be further exploited in genome-wide RNAi screens for insulator components. It provides a useful tool that complements the transgenic and genetic approaches for studying this important class of regulatory elements.
Highlights
Chromatin boundaries, known as insulators, regulate gene activity by organizing active and repressive chromatin domains and modulate enhancer-promoter interactions
The enhancer and promoter pair was combined with a GFP reporter in a P-element backbone and introduced into Drosophila s Line 2 (S2) cells via transient transfection
Fluorescence-Activated Cell Sorting (FACS) showed that this corresponded with a 10fold increase in the frequency of GFP positive cells when compared with the no-induction control
Summary
Known as insulators, regulate gene activity by organizing active and repressive chromatin domains and modulate enhancer-promoter interactions. The best-known examples of chromatin boundary elements include scs and scs', which delimit the active chromatin domain of the Drosophila hsp genes during heatshock [9,10]. Despite the diverse genomic contexts and different organismal origins, chromatin boundaries are characterized by either one or both of the following functional properties: their ability to block enhancer-promoter interactions when positioned interveningly (insulator activity, see [17,18,19,20,21,22]), and their ability to protect reporter genes from the transcriptional influences from the surrounding genome (barrier activity, [9,23,24,25]). The mechanism of boundary activity remains poorly understood This is partly due to our ignorance about their protein components, and a lack of systematic and comparative analyses of various insulator activities.
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