Engrailed cooperates directly with Extradenticle and Homothorax on a distinct class of homeodomain binding sites to repress sloppy paired

Abstract

Even skipped (Eve) and Engrailed (En) are homeodomain-containing transcriptional repressors with similar DNA binding specificities that are sequentially expressed in Drosophila embryos. The sloppy-paired (slp) locus is a target of repression by both Eve and En. At blastoderm, Eve is expressed in 7 stripes that restrict the posterior border of slp stripes, allowing engrailed (en) gene expression to be initiated in odd-numbered parasegments. En, in turn, prevents expansion of slp stripes after Eve is turned off. Prior studies showed that the two tandem slp transcription units are regulated by cis-regulatory modules (CRMs) with activities that overlap in space and time. An array of CRMs that generate 7 stripes at blastoderm, and later 14 stripes, surround slp1 (Fujioka and Jaynes, 2012). Surprisingly given their similarity in DNA binding specificity and function, responsiveness to ectopic Eve and En indicates that most of their direct target sites are either in distinct CRMs, or in different parts of coregulated CRMs. We localized cooperative binding sites for En, with the homeodomain-containing Hox cofactors Extradenticle (Exd) and Homothorax (Hth), within two CRMs that drive similar expression patterns. Functional analysis revealed two distinct, redundant sites within one CRM. The other CRM contains a single cooperative site that is both necessary and sufficient for repression in the en domain. Correlating in vivo and in vitro analysis suggests that cooperativity with Exd and Hth is a key ingredient in the mechanism of En-dependent repression, and that apparent affinity in vitro is an unreliable predictor of in vivo function.

Using competition assays to quantitatively model cooperative binding by transcription factors and other ligands

“…In a paper published in 2012 [38], Fujioka et al characterized several cooperative binding sites for the Engrailed (En) protein within the sloppy-paired ( slp ) locus of Drosophila. En shows strongly cooperative binding to each of these sites with a cofactor complex, which contains one molecule each of the proteins Exd and Hth.…”

“…However, an apparent paradox was uncovered in that the two lower affinity sites showed distinctly different functional characteristics in vivo , despite very similar behaviors in the binding assays. The functional assay involved repression of a reporter transgene in En-expressing cells in the developing Drosophila embryo, which is dependent on a functional binding site for En and Exd/Hth [38]. We speculated that the subtle differences we observed in their apparent cooperativity might be responsible for their distinct functional potencies: the more cooperative site gave more complete repression in vivo .…”

“…2 system plus unlabeled competitor, U AB , with its own cooperativity and equilibrium parameters. This system is used to describe 3 experiments [38], each with the same labeled DNA oligo (B1a), competing with an unlabeled oligo, either A2a, B1b, or B1a itself. Each experiment uses the same set of equations, but with different parameter values, chosen to approximate a situation encountered experimentally [38].…”

“…This provides the means to predict the binding behavior of cooperating proteins over the full range of concentrations. Motivated by recent identification and analysis of cooperative binding sites for Engrailed with its partner complex Extradenticle/Homothorax (Exd/Hth) [38], we devise a novel method for determining binding constants for individual ligands using competition assays [39], which we show has significant advantages over saturation binding assays. We go on to devise a method to find the cooperativity factor and the second equilibrium constant when only one of the two equilibrium constants can be accurately measured using single-ligand binding.…”

Genomic Strategies for Embryonic Development

“…Many individual enhancers active in this process have been beautifully analyzed. These regulatory modules read the dynamically changing ambient regulatory landscape so that when attached to reporters and introduced into the Drosophila germ line, they faithfully recreate the specific A/P or D/V patterns of expression of their particular parent genes, and a large literature on these continues to accumulate (for reviews see Schroeder et al, 2004;Davidson, 2006;Fujioka et al, 2012). The performance of these enhancers is in itself of enormous importance, because it demonstrates that the genomic cisregulatory code contains all the necessary spatial information.…”