Enhancer blocking by the Drosophila gypsy insulator depends upon insulatoranatomy and enhancer strength

Insulators are specialized DNA sequences that prevent enhancer-activated tr anscription only when interposed between an enhancer and its target promote r. The Drosophila gypsy retrotransposon contains an insulator composed of 1 2 degenerate binding sites for the Suppressor of Hairy-wing [Su(Hw)] protei n that are separated by AT-rich DNA possessing sequence motifs common to ma trix/scaffold attachment regions (MARs/SARs). To further understand mechani sms of insulator function, the parameters required for the gypsy insulator to prevent enhancer-activated transcription were examined. Synthetic bindin g regions were created by reiteration of a single Su(Hw) binding site that lacked the MAR/SAR motifs. These synthetic binding regions reconstituted in sulator activity, suggesting that the property of enhancer blocking may be distinct from matrix association. We found that the number and spacing of S u(Hw) binding sites within the gypsy insulator, as well as the strength of the enhancer to be blocked, were important determinants of insulator functi on. These results provide a link between transcription and insulation, sugg esting that these processes may be mechanistically interconnected.