Architectural protein pita cooperates with dCTCF in organization of functional boundaries in bithorax complex

Boundaries in the Bithorax complex (BX-C) of Drosophila delimit autonomous regulatory domains that drive parasegment-specific expression of homeotic genes. BX-C boundaries have two crucial functions: they must block crosstalk between adjacent regulatory domains and at the same time facilitate boundary bypass. The C2H2 zinc-finger protein Pita binds to several BX-C boundaries, including Fab-7 and Mcp. To study Pita functions, we have used a boundary replacement strategy by substituting modified DNAs for the Fab-7 boundary, which is located between the iab-6 and iab-7 regulatory domains. Multimerized Pita sites block iab-6↔iab-7 crosstalk but fail to support iab-6 regulation of Abd-B (bypass). In the case of Fab-7, we used a novel sensitized background to show that the two Pita-binding sites contribute to its boundary function. Although Mcp is from BX-C, it does not function appropriately when substituted for Fab-7: it blocks crosstalk but does not support bypass. Mutation of the Mcp Pita site disrupts blocking activity and also eliminates dCTCF binding. In contrast, mutation of the Mcp dCTCF site does not affect Pita binding, and this mutant boundary retains partial function.