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Significance ThreeHox genes—Ubx ,abd-A, andAbd-B —in theDrosophila bithorax complex are regulated by enhancer domains that drive parasegment-specific expression. The boundary/insulator elements flank the regulatory domains, ensuring their autonomous activity. The presence of boundaries poses a paradox of how enhancers located in regulatory domains can bypass intervening boundaries to properly regulate one of theHox genes. Here we constructed in vivo boundaries consisting of multimerized binding sites for several architectural proteins that block cross-talk between neighboring regulatory domains and do not allow enhancer bypass. However, adding the 242-bp core of the endogenous boundary,Fab-7 , completely restores bypass activity. Our results show that thebithorax boundary can be functionally divided into separate insulator and bypass modules.

Complete reconstitution of bypass and blocking functions in a minimal artificial Fab-7 insulator from Drosophila bithorax complex