Functional Requirements for Fab-7 Boundary Activity in the Bithorax Complex

Abstract

Chromatin boundaries are architectural elements that determine the three-dimensional folding of the chromatin fiber and organize the chromosome into independent units of genetic activity. The Fab-7 boundary from the Drosophila bithorax complex (BX-C) is required for the parasegment-specific expression of the Abd-B gene. We have used a replacement strategy to identify sequences that are necessary and sufficient for Fab-7 boundary function in the BX-C. Fab-7 boundary activity is known to depend on factors that are stage specific, and we describe a novel ∼700-kDa complex, the late boundary complex (LBC), that binds to Fab-7 sequences that have insulator functions in late embryos and adults. We show that the LBC is enriched in nuclear extracts from late, but not early, embryos and that it contains three insulator proteins, GAF, Mod(mdg4), and E(y)2. Its DNA binding properties are unusual in that it requires a minimal sequence of >65 bp; however, other than a GAGA motif, the three Fab-7 LBC recognition elements display few sequence similarities. Finally, we show that mutations which abrogate LBC binding in vitro inactivate the Fab-7 boundary in the BX-C.

Supplemental material for this article may be found at http://dx.doi.org/10.1128/MCB.00456-15.

ACKNOWLEDGMENTS

We thank Robert Maeda and Henrik Gyurkovics for insightful discussions. We also thank Eva Favre, Benjamin Barandun, Jorge Faustino, and Gordon Gray for excellent technical assistance. We thank Anton Golovnin, Pavel Georgiev, Elissa Lei, Victor Coreces, Carl Wu, and David Gilmour for the gift of antibodies. We also thank the reviewers for their thoughtful comments.

F.K. acknowledges support by grants from the Donation Claraz, the State of Geneva, and the Swiss National Fund for Research. P.S. acknowledges support from a grant from the NIH (GM043432) and from a grant to the Gene Biology Institute by the Russian Federation Ministry of Education and Science (14.B25.31.0022).