Abstract
BackgroundBoundaries in the Drosophila bithorax complex delimit autonomous regulatory domains that activate the parasegment (PS)-specific expression of homeotic genes. The Fab-7 boundary separates the iab-6 and iab-7 regulatory domains that control Abd-B expression in PS11 and PS12. This boundary is composed of multiple functionally redundant elements and has two key activities: it blocks crosstalk between iab-6 and iab-7 and facilitates boundary bypass.ResultsHere, we have used a structure–function approach to elucidate the biochemical properties and the in vivo activities of a conserved BEN domain protein, Insensitive, that is associated with Fab-7. Our biochemical studies indicate that in addition to the C-terminal BEN DNA-binding domain, Insv has two domains that mediate multimerization: one is a coiled-coil domain in the N-terminus, and the other is next to the BEN domain. These multimerization domains enable Insv to bind simultaneously to two canonical 8-bp recognition motifs, as well as to a ~ 100-bp non-canonical recognition sequence. They also mediate the assembly of higher-order multimers in the presence of DNA. Transgenic proteins lacking the N-terminal coiled-coil domain are compromised for boundary function in vivo. We also show that Insv interacts directly with CP190, a protein previously implicated in the boundary functions of several DNA-binding proteins, including Su(Hw) and dCTCF. While CP190 interaction is required for Insv binding to a subset of sites on polytene chromosomes, it has only a minor role in the boundary activity of Insv in the context of Fab-7.ConclusionsThe subdivision of eukaryotic chromosomes into discrete topological domains depends upon the pairing of boundary elements. In flies, pairing interactions are specific and typically orientation dependent. They occur in cis between neighboring heterologous boundaries, and in trans between homologous boundaries. One potential mechanism for ensuring pairing-interaction specificity is the use of sequence-specific DNA-binding proteins that can bind simultaneously with two or more recognition sequences. Our studies indicate that Insv can assemble into a multivalent DNA-binding complex and that the N-terminal Insv multimerization domain is critical for boundary function.
Highlights
Boundaries in the Drosophila bithorax complex delimit autonomous regulatory domains that activate the parasegment (PS)-specific expression of homeotic genes
While we have not made a careful comparison of the expression patterns later in development, we have found that the ubi rescue transgenes express Insv in salivary glands, while the protein encoded by the endogenous insv gene is not found in this tissue
We found that the coiled-coil deletion in ubi-insvd50-79 transgene, at most, only marginally rescues the insvdependent defects in boundary function in insv23B; Fab7GAGA1-5 mutant flies; Western blots showed that the expression of the mutant protein is reduced compared to the wild-type Insv expressed under the ubi promoter
Summary
Boundaries in the Drosophila bithorax complex delimit autonomous regulatory domains that activate the parasegment (PS)-specific expression of homeotic genes. Gene regulation in multicellular eukaryotes depends upon special elements called chromatin boundaries or insulators. These elements coordinate regulatory interactions with chromosome architecture by subdividing the chromosome into a series of functionally autonomous looped domains [1,2,3,4]. The subdivision of the chromosome into looped domains in flies depends upon boundary–boundary pairing interactions [1, 5,6,7,8] These pairing interactions are highly specific and typically orientation dependent. Unlike heterologous boundary pairing interactions, selfpairing interactions appear to be exclusively head-tohead. Both specificity and orientation dependence in pairing interactions are determined by the proteins associated with each element. In addition to the fly CTCF protein, several other fly polydactyl C2H2 zinc finger proteins are known to have insulator functions including Su(Hw), Pita, Zw5, Zipic, and Opbp [15,16,17,18]
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