Protein Domains Driving Hox Protein Function Figure 7. Mutually suppressive interaction of protein domains. A. Thoracic restricted expression of Dll (white arrows) followed by Dll enhancer driven b-gal (green) results from repression by AbdA (red) in the abdomen (upper panel). Ubiquitous AbdA expression driven by arm-Gal4 represses Dll thoracic expression (red arrows, middle panels). The effect of the AbdAHX/UA variants is illustrated (lower panels). Right panels are magnification of boxed thoracic areas. B. Increased thoracic Antp expression (green, white arrows) results from AbdA (red) repression in the abdomen (upper panels). Ubiquitous AbdA expression driven by arm-Gal4 represses Antp expression in the thorax (red arrows, middle panels). The effect of the AbdAHX/UA variants is illustrated (lower panels). Right panels are magnification of boxed thoracic areas. C. Abdominal segments are characterized by refringent denticles organized in a trapezoidal shape in segments A2 but not A1, while T2/T3 thoracic segments harbors thinner denticles (left panel). Upon AbdA thoracic expression driven by arm-Gal4, the first abdominal segment A1 and thoracic segments acquire abdominal features, including abdominal type of denticles, trapezoidal organization of denticles and suppression of a T1 specific feature (white arrow), the ‘‘beard’’ (middle panel). Full or intermediate transformations were observed for AbdA variants (see Text S1 for quantifying criteria). The effect of the AbdAHX/TD variants is illustrated (right panel). Weak A1 (wA1) stands for a transformation of thoracic denticles toward abdominal type of denticles, with an organization typical of A1, but with only a partial suppression of the beard in T1 (arrow). D. Snapshots from movies illustrating locomotion in wild type larvae (left panels), or in larvae expressing ubiquitously AbdA (middle panels) or AbdAHX/UA variant (right panels) driven by the arm-Gal4 driver. White boxed areas show the progression of a peristaltic waves in the abdomen. The red boxed area shows an ectopic peristaltic wave in the thorax following ectopic AbdA expression in the thorax. Graphs in A–D (% of remaining activities compared to the wild type AbdA protein (WT) following domain mutations) using the boxplot representation summarize quantitative analyses (see Text S1 and Figure S7 (Dll), S8 (Antp), and S9 (A2 epidermal morphology) for full illustration, and Figure S10 for data on larval locomotion experiments. A graded color-coded bar above the graphs illustrates the level of protein activity, ranging from light green (full activity) to black (no activity). doi:10.1371/journal.pgen.1002302.g007 depending on the cognate sequence [51]. Of note, a role for the target sequence in controlling the structure and activity of the glucocorticoid receptor has also been recently reported [52], indicating that this may generally apply for many DNA binding transcription factors. Mechanisms underlying the evolution of protein function Our results also have implication on how modifications in protein sequences are translated into changes in protein function during evolution. The HX domain, common to all Hox proteins, is ancient and found in all bilaterians, and provides a generic mode of PBC interaction (Figure 9). The UA domain, specific to some central Hox proteins (AbdA and Ubx in Drosophila), was acquired later, at the time of protostome/deuterostome radiation. It provides a distinct yet to be characterised PBC interaction mode, specific to some Hox paralogues only, allowing fine-tuning of Hox protein activity [22]. TD is found only in insect AbdA and not in Ubx proteins, suggesting that it arose after the duplication that generated Ubx and AbdA in the common ancestor of insects (Figure 9). Remarkably, within AbdA arthropod proteins, the HX domain has significantly diverged in some lineages like anopheles, while the TD domain has been strictly conserved. Conceptually, two non-exclusive models could account for the evolution of protein function following the acquisition of a novel protein domain. In the first one, the acquisition provides a novel molecular and functional property, which adds to pre-existing Figure 8. Hierarchical clustering of AbdA domain requirements. Hierarchical clustering of domain contribution reveals two functional modules and a predominant role for the UA domain. A graded color-coded bar above the graphs illustrates the level of protein activity, ranging from light green (full activity) to black (no activity). Neomorphic activity is depicted in red. Asterisks indicate Exd dependent biological readouts. Jacknife values are indicated for each node. doi:10.1371/journal.pgen.1002302.g008 PLoS Genetics | www.plosgenetics.org 10 October 2011 | Volume 7 | Issue 10 | e1002302