The <i>gypsy</i> insulators directly interact with the enhancer and promoter of the <i>white</i> gene.

<p>(A) Eye phenotypes in flies from the transgenic line heterozygous (P/+) for the construct and in flies after deletion of either the proximal <i>gypsy</i> insulator, or both insulators, or introduction of the <i>mod(mdg4)^u1</i> mutation. (B) The 3C profile of the E(Gy)^R_4.5W(Gy) line and its derivatives analyzed at the pupa stage for an anchor fragment located in the enhancer or promoter region or downstream of the distal <i>gypsy</i> insulator. The transgene and the surrounding genomic region are drawn to scale. Relative interaction frequencies between <i>Dpn</i>II restriction fragments selected for this analysis (gray vertical bands) and anchor regions (purple vertical bands) are shown for the construct (blue-square line), and its derivatives with deletion of proximal <i>gypsy</i> (green-circle line), proximal <i>gypsy</i> at the <i>mod(mdg4)^u1</i> background (yellow-triangle line), and both <i>gypsy</i> copies (red-cross line). Samples were normalized by qPCR relative to an undigested locus. Error bars show standard deviations from the means of three independent experiments. Transgene DNA is shown as blue line at the bottom of figure, and the boundaries of the transgenes are indicated by blue triangles. The <i>white</i> gene, eye enhancer, and <i>gypsy</i> insulator are represented, respectively, by yellow and green boxes and red pentagon. The lox and frt sites allowing the excision of insulators are indicated by vertical black brackets. Other designations are as in <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1003606#pgen-1003606-g001" target="_blank">Figure 1</a>.