Complete reconstitution of bypass and blocking functions in a minimal artificial <i>Fab-7</i> insulator from <i>Drosophila bithorax</i> complex.

Abstract

Boundaries in the <i>bithorax</i> complex (BX-C) delimit autonomous regulatory domains that drive parasegment-specific expression of the <i>Hox</i> genes <i>Ubx</i>, <i>abd-A,</i> and <i>Abd-B</i> The <i>Fab-7</i> boundary is located between the <i>iab-6</i> and <i>iab-7</i> domains and has two key functions: blocking cross-talk between these domains and at the same time promoting communication (boundary bypass) between <i>iab-6</i> and the <i>Abd-B</i> promoter. Using a replacement strategy, we found that multimerized binding sites for the architectural proteins Pita, Su(Hw), and dCTCF function as conventional insulators and block cross-talk between the <i>iab-6</i> and <i>iab-7</i> domains; however, they lack bypass activity, and <i>iab-6</i> is unable to regulate <i>Abd-B</i> Here we show that an ∼200-bp sequence of dHS1 from the <i>Fab-7</i> boundary rescues the bypass defects of these multimerized binding sites. The dHS1 sequence is bound in embryos by a large multiprotein complex, Late Boundary Complex (LBC), that contains the zinc finger proteins CLAMP and GAF. Using deletions and mutations in critical GAGAG motifs, we show that bypass activity correlates with the efficiency of recruitment of LBC components CLAMP and GAF to the artificial boundary. These results indicate that LBC orchestrates long-distance communication between the <i>iab-6</i> regulatory domain and the <i>Abd-B</i> gene, while the Pita, Su(Hw), and dCTCF proteins function to block local cross-talk between the neighboring regulatory domains <i>iab-6</i> and <i>iab-7</i>.