Insights into Hox Protein Function from a Large Scale Combinatorial Analysis of Protein Domains Samir Merabet1,2.*, Isma Litim-Mecheri1,2., Daniel Karlsson 3 , Richa Dixit4, Mehdi Saadaoui1,2, Bruno Monier1,2, Christine Brun2,5,6, Stefan Thor3, K. Vijayraghavan4, Laurent Perrin1,2, Jacques Pradel1,2, Yacine Graba1,2* 1 Institut de Biologie du De´veloppement de Marseille Luminy, IBDML, UMR6216 CNRS, Parc Scientifique de Luminy, Case 907, Marseille, France, 2 Universite´ de la Me´diterrane´e, Parc Scientifique de Luminy, Marseille, France, 3 Department of Clinical and Experimental Medicine, Linkoping University, Linkoping, Sweden, 4 National Centre for Biological Sciences, Tata Institute of Fundamental Research, Bangalore, India, 5 TAGC, U928 Inserm, Parc Scientifique de Luminy, Case 928, Marseille, France, 6 CNRS, Marseille, France Abstract Protein function is encoded within protein sequence and protein domains. However, how protein domains cooperate within a protein to modulate overall activity and how this impacts functional diversification at the molecular and organism levels remains largely unaddressed. Focusing on three domains of the central class Drosophila Hox transcription factor AbdominalA (AbdA), we used combinatorial domain mutations and most known AbdA developmental functions as biological readouts to investigate how protein domains collectively shape protein activity. The results uncover redundancy, interactivity, and multifunctionality of protein domains as salient features underlying overall AbdA protein activity, providing means to apprehend functional diversity and accounting for the robustness of Hox-controlled developmental programs. Importantly, the results highlight context-dependency in protein domain usage and interaction, allowing major modifications in domains to be tolerated without general functional loss. The non-pleoitropic effect of domain mutation suggests that protein modification may contribute more broadly to molecular changes underlying morphological diversification during evolution, so far thought to rely largely on modification in gene cis-regulatory sequences. Citation: Merabet S, Litim-Mecheri I, Karlsson D, Dixit R, Saadaoui M, et al. (2011) Insights into Hox Protein Function from a Large Scale Combinatorial Analysis of Protein Domains. PLoS Genet 7(10): e1002302. doi:10.1371/journal.pgen.1002302 Editor: Artyom Kopp, University of California Davis, United States of America Received May 25, 2011; Accepted July 24, 2011; Published October 27, 2011 Copyright: 2011 Merabet et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Funding: Work in the YG and JP laboratory is supported by the CNRS, Universite´ de la Me´diterrane´e, and grants from ANR, FRM, CEFIPRA, and ARC; work in the ST laboratory is supported by the Swedish Research Council, the Swedish Strategic Research Foundation, the Knut and Alice Wallenberg Foundation, the Swedish ‘‘Hja¨rnfonden,’’ ‘‘Cancerfonden,’’ and the Swedish Royal Academy of Sciences; work in the LP laboratory is supported by the CNRS, Universite´ de la Me´diterranne´e, and grants from ANR and AFM. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Competing Interests: The authors have declared that no competing interests exist. * E-mail: yacine.graba@univmed.fr (YG); samir.merabet@univmed.fr (SM) . These authors contributed equally to this work. Introduction How the diversity of animal body plans is established remains a central question in developmental and evolutionary biology [1,2]. A key step towards understanding the molecular basis underlying diversity is to decipher mechanisms controlling proper genome expression, and how variations in these mechanisms have been at the origin of developmental and evolutionary diversity. While a large number of studies have focused on the impact of cis-regulatory sequences organization (reviewed in [3]), deciphering the intrinsic functional organization of trans-acting transcription factors remains largely unaddressed. Studies have identified functional domains ([4– 9] and [7,10,11] for reviews), but how different protein domains jointly and collectively act for defining the overall activity has been poorly assessed. Yet, a recent study highlights that the synthetic shuffling of protein domains within proteins of the yeast-mating signaling pathway results in the diversification of the mating behavior, demonstrating the importance of protein domain interactions for functional diversification [12]. Hox genes, which encode homeodomain (HD)-containing transcription factors, provide a suitable paradigm to decipher how function is encoded within protein sequence, and how associated changes may constitute the origin of functional specification and diversification. Hox genes have arisen from duplication events of ancestral genes, followed by sequence divergence that promoted the emergence of up to 14 paralogous groups in vertebrates. Hox paralogue proteins display distinct regulatory functions, promoting axial morphological diversifica- tion in all bilaterian animals [13–17]. Previous work has established that sequence changes in the HD, the DNA binding domain, and a few additional protein domains, have played a major role in the diversification of Hox protein function [4–9,18– 21]. However, how protein domains functionally interact to shape overall protein activity remains elusive. We focused on three protein domains from the Drosophila central Hox paralogue protein Abdominal (AbdA, Figure 1). These domains are related by their demonstrated or potential involve- ment in the recruitment of the Extradenticle (Exd) cofactor, homologous to vertebrate PBX proteins, known to have key roles in establishing Hox functional specificity. The first domain, known as hexapeptide (HX) or PID (Pbx Interacting Domain), with a core YPWM sequence, is found in all Hox paralogue groups, with the PLoS Genetics | www.plosgenetics.org 1 October 2011 | Volume 7 | Issue 10 | e1002302