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Proc Natl Acad Sci U S A. 2019 Jul 2;116(27):13462-13467. doi: 10.1073/pnas.1907190116. Epub 2019 Jun 17.

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

Author information

1
Department of the Control of Genetic Processes, Institute of Gene Biology, Russian Academy of Sciences, 119334 Moscow, Russia.
2
Department of Molecular Biology, Princeton University, Princeton, NJ 08544.
3
Department of Molecular Biology, Princeton University, Princeton, NJ 08544; pschedl@princeton.edu georgiev_p@mail.ru.
4
Laboratory of Gene Expression Regulation in Development, Institute of Gene Biology, Russian Academy of Sciences, 119334 Moscow, Russia.
5
Department of the Control of Genetic Processes, Institute of Gene Biology, Russian Academy of Sciences, 119334 Moscow, Russia; pschedl@princeton.edu georgiev_p@mail.ru.

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.

KEYWORDS:

CLAMP; LBC; architectural proteins; chromatin insulator; distant interactions

PMID:
31209019
PMCID:
PMC6613175
[Available on 2019-12-17]
DOI:
10.1073/pnas.1907190116

Conflict of interest statement

The authors declare no conflict of interest.

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