Despite tremendous body form diversity in nature, bilaterian animals share common sets of developmental genes that display conserved expression patterns in the embryo. Among them are the Hox genes, which define different identities along the anterior–posterior axis. Hox proteins exert their function by interaction with TALE transcription factors. Hox and TALE members are also present in some but not all non-bilaterian phyla, raising the question of how Hox–TALE interactions evolved to provide positional information. By using proteins from unicellular and multicellular lineages, we showed that these networks emerged from an ancestral generic motif present in Hox and other related protein families. Interestingly, Hox-TALE networks experienced additional and extensive molecular innovations that were likely crucial for differentiating Hox functions along body plans. Together our results highlight how homeobox gene families evolved during eukaryote evolution to eventually constitute a major patterning system in Eumetazoans.

Homeodomain proteins are evolutionary conserved proteins present in the entire eukaryote kingdom. They execute functions that are essential for life, both in developing and adult organisms. Most homeodomain proteins act as transcription factors and bind DNA to control the activity of other genes. In contrast to their similar DNA binding specificity, homeodomain proteins execute highly diverse and context-dependent functions. Several factors, including genome accessibility, DNA shape, combinatorial binding and the ability to interact with many transcriptional partners, diversify the activity of homeodomain proteins and culminate in the activation of highly dynamic, context-specific transcriptional programs. Clarifying how homeodomain transcription factors work is central to our understanding of development, disease and evolution.

Hox proteins are well-established developmental regulators that coordinate cell fate and morphogenesis throughout embryogenesis. In contrast, our knowledge of their specific molecular modes of action is limited to the interaction with few cofactors. Here, we show that Hox proteins are able to interact with a wide range of transcription factors in the live Drosophila embryo. In this context, specificity relies on a versatile usage of conserved short linear motifs (SLiMs), which, surprisingly, often restrains the interaction potential of Hox proteins. This novel buffering activity of SLiMs was observed in different tissues and found in Hox proteins from cnidarian to mouse species. Although these interactions remain to be analysed in the context of endogenous Hox regulatory activities, our observations challenge the traditional role assigned to SLiMs and provide an alternative concept to explain how Hox interactome specificity could be achieved during the embryonic development.

HOX proteins achieve numerous functions by interacting with the TALE class PBX and MEIS cofactors. In contrast to this established partnership in development and disease, how HOX proteins could interact with PBX and MEIS remains unclear. Here, we present a systematic analysis of HOX/PBX/MEIS interaction properties, scanning all paralog groups with human and mouse HOX proteins in vitro and in live cells. We demonstrate that a previously characterized HOX protein motif known to be critical for HOX-PBX interactions becomes dispensable in the presence of MEIS in all except the two most anterior paralog groups. We further identify paralog-specific TALE-binding sites that are used in a highly context-dependent manner. One of these binding sites is involved in the proliferative activity of HOXA7 in breast cancer cells. Together these findings reveal an extraordinary level of interaction flexibility between HOX proteins and their major class of developmental cofactors.