Architectural protein Pita cooperates with dCTCF in organization of functional boundaries in Bithorax complex

Abstract

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.

The BEN Domain Protein Insensitive Binds to the Fab-7 Chromatin Boundary To Establish Proper Segmental Identity in Drosophila

“…200 bp and their activities depend upon unique assemblages of associated proteins. In the cases that have been studied in detail, the cisand trans-acting elements are often functionally redundant with no single element being absolutely essential (Cuartero et al 2014;Zolotarev et al 2016;Kyrchanova et al 2017). This means that a mutation in the recognition sequence for a boundary protein or in the protein itself may have no obvious phenotypic consequences.…”

“…Elba and LBC are not the only factors that bind to Fab-7 boundary sequences. The constitutively expressed C2H2 zinc finger protein Pita binds to two sites in HS2 (Maksimenko et al 2015;Kyrchanova et al 2017). Because of functional redundancy, mutations in these Pita sites in a Fab-7 boundary replacement that has HS* + HS1 + HS2 (but not HS3) have no effect on boundary function.…”

Boundaries support specific long-distance interactions between enhancers and promoters inDrosophila Bithoraxcomplex

“…Adult abdominal cuticles of homozygous enclosed 3-4 day old flies were prepared essentially as described in (Kyrchanova et al 2017) and mounted in 100% glycerol. Photographs in the bright or dark field were taken on the Nikon SMZ18 stereomicroscope using Nikon DS-Ri2 digital camera, processed with ImageJ 1.50c4 and Fiji bundle 2.0.0-rc- dsRed gene was using for selection of flies with iab-4 deletion.…”

“…For example, the iab-6 regulatory domain must "jump over" or "bypass" Fab-7 and Fab-8 in order to interact with the Abd-B promoter ( Fig 1A). That the blocking function of boundaries could pose a significant problem has been demonstrated by experiments in which Fab-7 is replaced by heterologous elements such as scs, gypsy or multimerized binding sites for the architectural proteins dCTCF, Pita or Su(Hw) [26,[29][30][31].…”

“…However, several findings argue against this notion. For one, BX-C boundaries resemble those elsewhere in the genome in that they contain binding sites for architectural proteins such as Pita, dCTCF, and Su(Hw) [24,[31][32][33][34][35]. Consistent with their utilization of these generic architectural proteins, when placed between enhancers (or silencers) and a reporter gene, BX-C boundaries block regulatory interactions just like boundaries from elsewhere in the genome [20,[36][37][38][39][40][41][42].…”