Elsevier

Developmental Biology

Volume 366, Issue 2, 15 June 2012, Pages 382-392
Developmental Biology

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

https://doi.org/10.1016/j.ydbio.2012.04.004Get rights and content
Under an Elsevier user license
open archive

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.

Highlights

► Multiple slp CRMs are targets of repression by both Engrailed and Even-skipped. ► Despite similar individual binding specificities, they have distinct target sites. ► Engrailed binds with the Hox cofactors Exd and Hth to sites in two separate CRMs. ► Several essential target sites were localized and their functionalities compared. ► Affinity in vitro, by itself, poorly predicts functionality in vivo.

Keywords

Homeodomain protein binding
Cooperative homeoprotein sites
Engrailed
Extradenticle
Homothorax
Sloppy paired

Cited by (0)