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

► 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.