Transcription factor paralogs orchestrate alternative gene regulatory networks by context-dependent cooperation with multiple cofactors

In eukaryotes, members of large transcription factor families often exhibit similar DNA binding properties in vitro , yet initiate paralog-specific gene regulatory networks in vivo . The serially homologous first (T1) and third (T3) thoracic legs of Drosophila , which result from alternative gene regulatory networks specified by the Hox proteins Scr and Ubx, respectively, offer a unique opportunity to address this paradox in vivo . Genome-wide analyses using epitope-tagged alleles of both Hox loci in the T1 and T3 leg imaginal discs, which are the precursors to the adult appendages and ventral body regions, show that ∼8% of Hox binding is paralog-specific. Binding specificity is mediated by interactions with distinct cofactors in different domains: the known Hox cofactor Exd acts in the proximal domain and is necessary for Scr to bind many of its paralog-specific targets, while in the distal leg domain, we identified the homeodomain protein Distal-less (Dll) as a novel Hox cofactor that enhances Scr binding to a different subset of genomic loci. Reporter genes confirm the in vivo roles of Scr+Dll and suggest that ∼1/3 of paralog-specific Hox binding in enhancers is functional. Together, these findings provide a genome-wide view of how Hox paralogs, and perhaps paralogs of other transcription factor families, orchestrate alternative downstream gene networks and suggest the importance of multiple, context-specific cofactors.