Complete reconstitution of bypass and blocking functions in a minimal artificialFab-7insulator fromDrosophila bithoraxcomplex

Abstract

Boundaries in the bithorax complex (BX-C) delimit autonomous regulatory domains that drive parasegment-specific expression of the Hox genes Ubx, abd-A, and Abd-B. The Fab-7 boundary is located between the iab-6 and iab-7 domains and has two key functions: blocking cross-talk between these domains and at the same time promoting communication (boundary bypass) between iab-6 and the Abd-B 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 iab-6 and iab-7 domains; however, they lack bypass activity, and iab-6 is unable to regulate Abd-B. Here we show that an ∼200-bp sequence of dHS1 from the Fab-7 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 iab-6 regulatory domain and the Abd-B gene, while the Pita, Su(Hw), and dCTCF proteins function to block local cross-talk between the neighboring regulatory domains iab-6 and iab-7.

The insulator functions of theDrosophilapolydactyl C2H2 zinc finger protein CTCF: Necessity versus sufficiency

“…This deletion removes the recognition sequence for the LBC but retains the two dCTCF-binding sites. As previously reported (19), F8 209 blocks cross-talk between iab-6 and iab-7, similar to F8 337 ; however, it does not support bypass ( Fig. 2A).…”

“…Although the two dCTCF sites in Fab-8 alone are not sufficient to block cross-talk between iab-6 and iab-7, we found that a multimer containing four dCTCF (CTCF ×4 ) sites displayed near-complete blocking activity (19). To determine the minimal number of dCTCF sites necessary to block cross-talk between iab-6 and iab-7 activity, we generated a CTCF ×3 replacement ( Fig.…”

“…on September 30, 2020 http://advances.sciencemag.org/ Downloaded from one or more of the Abd-B boundaries (e.g., iab-6 and Fab-7/Fab-8) can "jump over" the intervening boundaries and contact the Abd-B promoter (28). In the case of Fab-8, blocking activity depends on two dCTCF-binding sites, whereas bypass activity requires recognition sequences for a large multiprotein complex, called the large boundary complex (LBC) (19). Similar to the deletions of Fab-7 and Fab-8, deletions of Fab-6 (which separates iab-5 and iab-6), Mcp (which separates iab-4 and iab-5), and Fub (which separates bxd/pbx and iab-2) result in GOF transformations in the parasegment (segment) specified by the centromere proximal regulatory domain (22,24,25).…”

Distinct Elements Confer the Blocking and Bypass Functions of the Bithorax Fab-8 Boundary

“…On the other hand, the proximal Fab-8 sequences are needed for the bypass activity of the Fab-8 boundary. Since we have found that dHS1 has bypass activity when combined with multimerized dCTCF or Pita sites (Kyrchanova et al 2019), we wondered whether dHS1 would be able to substitute for the proximal region of Fab-8 and rescue the bypass defects of F8 . To test this, we combined dHS1 with F8 129-337 .…”

“…To interact productively with the Abd-Bm promoter, these regulatory domains must be able to circumvent the blocking activity of the intervening boundary elements. That the presence of these intervening boundaries is problematic was illustrated by experiments in which Fab-7 was replaced by a series of heterologous boundaries including scs, Mcp, and multimerized binding sites for the chromosome architectural proteins CCCTC-binding factor (dCTCF) and Pita (Hogga et al 2001;Iampietro et al 2008;Kyrchanova et al 2016Kyrchanova et al , 2019. In all of these cases, the heterologous boundaries were able to block cross talk between iab-6 and iab-7 just like Fab-7; however, they failed to support bypass.…”