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 ~70% 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. Overall design: RNA-seq (3 biological replicates) was used to characterize the transcriptomes of T1, T2 and T3 leg discs. ATAC-seq (2 biological replicates) was used to profile chromatin accessibility in T1, T2 and T3 leg discs. The 3xFLAG tag was inserted into the endogenous Hox genes Scr and Ubx. A 3xFLAG-Scr(YPWM*) allele, which encodes a mutant 3xFLAG-Scr protein unable to interact with the Hox cofactor Exd, was also generated. ChIP-seq (2 biological replicates) against various 3xFLAG-tagged Hox proteins was performed, and the wild type animals, which lacks the 3xFLAG tag, was used as negative control. ChIP-seq was also performed against the homeodomain TF Dll. Gel-free Selex-seq was used to characterize the binding preferences of selected TF monomers and heterodimers.