Protein Domains Driving Hox Protein Function tube are composed of six cardiac cells, labeled in blue by Mef2. The two most posterior cells express Doc1 (green). The thoracic hemi-segments lack the anterior Doc1 positive cells. This distinction between thoracic and abdominal segments is not affected following maternal and zygotic loss of exd. doi:10.1371/journal.pgen.1002302.g006 zygotic loss [49]. The absence of peristaltic waves in this genetic context indicates a strict requirement of Exd for abdominal peristalsis (Figure S10). Taken together with the protein domain requirement results, the exd dependency indicates that the HX, UA and TD domains, known (HX and UA) or candidate (TD) Exd recruiting domains, are also required for Exd-independent function. This is supported by the HX/UA requirement for heart lineage specification, by the HX and UA requirement for proper regulation of the dpp target gene, the HX/TD requirement for Antp repression and the requirement of TD for dpp target regulation. Collectively, this highlights that the HX and UA (and likely TD) protein domains are multifunctional, serving in some biological context Exd interaction function, while in others, they are used differently, for a molecular activity that still remains to be defined. Hierarchical clustering reveals two functional modules and a predominant role for the UA domain The complete set of quantitative data was analyzed using a hierarchical clustering method (Figure 8; see Materials and Methods). Clustering according to biological readouts does not reveal any clear grouping, regarding for instance developmental stage or tissue type, suggesting that the forces that govern domain usage and interaction between protein domains mostly reside in the regulated target gene. By contrast, clustering according to protein domains clearly reveals a hierarchical requirement of the domains for the various AbdA functions analyzed here. A bipartition of AbdA variants is observed, with the mutants for the HX, the TD and HX/TD domains on the one hand, and variants mutant for the UA domain, alone or in combination, on the other hand. Such bipartition suggests the existence of two functional modules that can be distinguished based on UA domain requirement. The first module, which relies mostly on the HX and TD domains, is used for a small subset of AbdA functions only. The second module relies on the activity of the HX, TD and UA domains, yet the requirements of the HX and TD domains are revealed only in UA deficient context. Thus, the driving force in this second functional module is the UA domain, as its mutation unmasks the requirement for the HX and TD domains, which is not revealed by their single or combined mutations. These results identify a prominent role of the UA domain in AbdA function. Discussion A different complementary approach to Hox protein function Studies towards deciphering the mode of action of Hox proteins have so far essentially concentrated on how individual protein domains contribute to protein function. These focused approaches allowed in depth analyses, unraveling the intimate molecular and sometimes structural details of how protein domains contribute to protein function, providing decisive insights into how Hox proteins reach specificity. This work provides a different complementary approach towards deciphering the mode of action of Hox proteins. First it aims at studying protein domains in combinations, using combined and not only single protein domain mutations, considering that the overall protein activity is likely not a sum of the activity of individual protein domains, and that novel properties may emerge from interactions between protein domains. Second, it uses extensive in vivo biological readout, (most of the known AbdA functions), instead of a single or a few functions. While impairing the in depth analyses of previous focused approaches, the large functional window covered by this study allows the identification of features underlying the intrinsic functional organization of the Hox protein AbdA. Although the approach taken relies on a gain of function strategy, special care was taken to select experimental conditions where proteins were expressed closed to physiological levels of expression. Biological readouts considered are functions that AbdA can sustain in ectopic places, suggesting that availability of AbdA protein partners is not a limitation of the experimental strategy chosen. Finally, the effects of expressing the AbdA variants (in all eleven biological readouts) were scored in regions anterior to the endogenous AbdA expression domain (ie in cells where the endogenous wild type gene product is not present), avoiding any further complexity that may result from competition with the endogenous AbdA protein. Below, we summarize how results obtained shed light on the mode of action of the Hox protein AbdA and discuss the evolutionary implications. Functional attributes and mode of protein domain usage: Implication for robustness and diversity This study identifies salient features underlying the intrinsic functional organization of the AbdA Hox transcription factor. Protein domains often display functional redundancy, with strong effects in most cases requiring simultaneous mutations of two or three domains. Redundancy was frequently observed between the HX and UA domains, or between the TD and UA domains, while redundancy between the HX and TD domains is less frequent (Figure 8). This indicates that redundancy does not necessarily rely on functional compensation through structurally related domains, since the HX and TD are closely related domains, while the UA domain is completely unrelated. Thus, functional redundancy rather reflects the potential to perform similar activities through distinct molecular strategies. This property likely confers robust- ness to Hox protein activity, accommodating mutations in protein domains without generally impacting on regulatory activities. Protein domains within AbdA also generally do not act as independent functional modules, but instead display a high degree of interactivity, as demonstrated by the non-additive effects of domain mutations in the majority of the biological readouts studied. In addition, protein domains are often multifunctional, in the sense that they serve different molecular functions. This is illustrated by the fact that the HX and UA domains, previously described to mediate Exd recruitment, are also required for Exd- independent processes. Thus domain interactivity and multi- functionality are hallmarks of AbdA regulatory activity. These properties provide means to apprehend the bases underlying Hox functional diversity with a restricted number of functional modules, and therefore may account for the variety of Hox- controlled biological functions. Protein domain usage and interaction between protein domains in AbdA strongly depends on the biological readout, suggesting that domain usage largely depends on the regulated target gene, and hence on the identity of the gene cis regulatory sequences. Recent reports support that DNA sequences impact on Hox protein activity: Hox binding site neighboring sequences are important for proper regulation of the reaper downstream target [50]; Sex combs reduced changes its conformation and activity PLoS Genetics | www.plosgenetics.org 8 October 2011 | Volume 7 | Issue 10 | e1002302