Author Summary Proteins perform essential regulatory functions, including control of gene transcription, a process central to development, evolution, and disease. While protein domains important for protein activity have been identi- fied, how they act together to define the activity of a protein remains poorly explored. The predominant view influenced by prokaryotic transcription factors is that protein domains constitute independent functional mod- ules, required for all aspects of protein activity. In this study, we used Hox proteins, evolutionarily conserved transcription factors playing key roles in the establishment of animal body plans, to examine how protein domains collectively shape protein activity. Results obtained using a broad range of biological readouts highlight a context- dependency in protein domain usage and interaction, revealing that protein domains are non-pleoitropic in nature. This suggests that protein modification may contribute more broadly to molecular changes underlying morphological diversity, so far thought to rely largely on modification of gene cis-regulatory sequences. exception of some posterior Hox proteins. Biochemical, structural and functional studies have shown that this motif mediates interaction with the Exd/PBX class of Hox cofactors (collectively referred as PBC). The second domain, termed UbdA (UA) is specifically found in the central Hox proteins AbdA and Ultrabithorax (Ubx). This paralogue-specific domain was recently shown to be required for Exd recruitment in the repression of the limb-promoting gene Distalless (Dll) [8,22]. The third domain (TD), similar in sequence (TDWM) to the YPWM motif, is also paralogue-specific. The TD motif retains the W that provides strong contact with the PBC class proteins, and matches the sequence of the HX motif in some Hox proteins (eg., Hoxa1). Evidence for an Exd recruiting role of the TD domain in AbdA however remains to be demonstrated. To start unraveling how protein domains collectively shape Hox protein activity, the effect of single, combined double or triple domain mutations were analyzed using most known AbdA functions as biological readouts. The large functional window covered by the study allows identifying functional attributes of protein domains taken in isolation and collectively, and a quantitative analysis by hierarchical clustering highlights the functional organization of the Hox protein AbdA. Given the phylogeny of the studied protein domains, the work has also implication regarding the mechanisms underlying the evolution of AbdA protein function. Results Expression of AbdA variants and biological readouts AbdA variants bearing single or all possible combinations of protein domain mutations (Figure 1A) were ectopically expressed through the binary UAS-Gal4 expression system [23]. Protein levels following induced expression were quantified and experimental conditions ensuring levels close to that of endogenous AbdA were selected (see Materials and Methods). Impact of AbdA variants on target gene control, phenotypic traits and locomotion behavior (Figure 1B), covering AbdA functions of increasing complexity in different tissues, were evaluated in the anterior region where the endogenous AbdA protein is absent. Quantified results (see Text S1) are presented as loss (and in few cases as gain) of regulatory potential. Eleven functional assays were used to assess domain requirements for AbdA activity (Figure 1B). Four assays rely on the regulation of Protein Domains Driving Hox Protein Function Figure 1. Combinatorial analyses of AbdA protein domains using a variety of biological readouts. A. AbdA protein variants generated. Crosses indicate domain mutations. B. Biological readouts used in this study. doi:10.1371/journal.pgen.1002302.g001 AbdA target genes, for which evidence of a direct regulation has been previously reported, including the regulation of Distalless (Dll) [8,24,25] and Antennapedia (Antp) [26] in the epidermis, and the regulation of wingless (wg) [27] and decapentaplegic (dpp) [28,29] in the visceral mesoderm. Six assays rely on analysis of phenotypic traits. One of these phenotypic trait, oenocyte specification, results from the regulation of a single target gene [30]. Others, cerebral branch [31], somatic muscles [32], A2 epidermal morphology [33,34], neuroblast [35,36] and heart cell lineage specification [37] likely depend of the coordinated regulation of several target genes. Finally, we also used a behavioral trait, larval locomotion, thought to rely on integrated AbdA function in two distinct tissues, the somatic musculature and the nervous system [38]. Dispensability of the HX, TD, and UA protein domains for somatic muscle specification In the somatic musculature, the abdominal specific pattern is characterized by the presence of muscle located ventrally and PLoS Genetics | www.plosgenetics.org 2 October 2011 | Volume 7 | Issue 10 | e1002302