Fig. 2. The identification of the Da REP domain reveals its role in modulating Twi activity in vitro.

(A) Schematic representation of the Da/E family of proteins. Activation domain 1 (AD1), activation domain 2 (AD2) (blue boxes), the basic helix-loop-helix (bHLH) region (orange box), and the REP domain, identified by homology mapping, are shown. (B) Sequence alignment of the REP domain of Da and vertebrate E proteins. Sequences shown include E2A (mouse), XE12 (Xenopus), ZE12 (Zebrafish), E2–2 (Human) and Da (Drosophila). Conserved amino acid residues are highlighted by blue boxes and asterisks. Highly conserved residues between Da and vertebrate species are denoted by two dots. Those with lower conservation are marked with one dot. Sequences were aligned using the ClustalW alignment algorithm (Thompson et al., 1994). (C) Schematic representation of the Da^Δ deletion construct. Amino acids 495–529 have been removed from Da. (D) Deletion of the Da REP domain modulates Twi activity. S2 cells were transfected with actin-LacZ plasmid, 175 bp Dmef2 enhancer-luciferase reporter plasmid, and various combinations of twi, da, da^Δ, twi-da, or twi-da^Δ expression vectors see Materials and Methods; Castanon et al., 2001). Luciferase activity is shown relative to control levels and error bars represent the standard error of the means of triplicate experiments. Cells transfected with twi (blue bar) show the highest level of reporter activation, whereas da and da^Δ, (yellow and orange, respectively) show similarly low activation of the Dmef2 enhancer. Cotransfection of twi and da (light green bar) caused minimal activation. twi and da^Δ cotransfection (dark green bar) results in high reporter gene transactivation. Transfection of twi-da and twi-da^Δ linked dimers (red and maroon bars, respectively) mirror the effects observed when twi and da, or twi and da^Δ, respectively, are cotransfected.